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Dapic kx – 922v how to reset

Dapic kx – 922v how to reset





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The van’t Hoff enthalpy estimates from ref. A copy of the annual report of the Company Dapic the fiscal year ended February 29,has been mailed to you with this proxy statement. Webba da Silva M. SVD Reset used to analyze temperature dependent circular dichroic spectra and to show that quadruplex denaturation was not a simple two-state process. Each member of the committee must be financially literate and at least one member of the committee must have accounting or related financial management expertise, as the Board interprets such qualifications in its business judgment. The assumption that metal binding does not in itself affect the absorption coefficients of the nucleotides is for illustrative purposes only and represents the simplest case. Experimentally, only four actual structures have How determined for the human telomere repeat; the 922v two topologies that have been determined were for different sequences.
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dapic kx - 922v how to reset

dapic kx - 922v how to reset

dapic kx - 922v how to reset

dapic kx - 922v how to reset

dapic kx - 922v how to reset

dapic kx - 922v how to reset

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Their studies indicated that the rate-limiting step in tetrameric quadruplex formation is a slow association of a pair of dimers that exist in relatively rapid equilibrium with single-stranded monomers. However, this structure is at best marginally stable at physiological pH and temperature though the actual state of protonation of the cytosines in the nucleus is far from clear. Please sign exactly as your name s appear on the Proxy. These considerations are discussed in greater detail below. However, in conclusion, it is worth repeating the classical caveat of kinetics: Sugimoto, have used crowding conditions in vitro to determine the influence on conformation and stability.

Specifically, for a single-step unimolecular reaction the relaxation rate constant is simply the sum of the forward and reverse reaction rate constants. The approach is applicable in the neighborhood of the T m and provides a direct measure of the folding and unfolding rate at a given temperature.

This approach was used in conjunction with hysteresis analysis to measure association and dissociation kinetics of the Oxytricha intramolecular quadruplex It was shown that the unfolding rate constant was extremely small, and that the association was characterized by negative apparent activation energy.

This latter requires a complex multistep process, such as the well-known zippering mechanism common in DNA duplex folding Stopped flow has a long history in enzyme kinetics, but has not been used for NA folding kinetics.

The titrations showed that folding is cooperative with respect to [cation] with Hill coefficients of 1. Cation half saturation concentrations for folding were 0. SVD of the wavelength dependence of the binding isotherms indicated that folding generally proceeds for ODN1 and ODN2 through at least one intermediate according to the Scheme The kinetics of folding was investigated using multi-wavelength stopped flow spectrophotometry The folding rates also tended to decrease with increasing temperature.

The following describes a possible mechanism that incorporates elementary steps from previous studies of oligonucleotide folding with the known structures of the final state and that is consistent with the kinetic data.

For a polynucleotide initially in the absence of cations, the first step may consist of rapid neutralization of backbone charge by added cation, resulting in collapse of extended polynucleotide to give an ensemble of compact hairpin structures.

Since the rate of complex formation depends on the fourth power of the monomer concentration, the actual time required for tetramerization is strongly dependent on the initial concentration of monomers and the nature of the cation and can vary from a few minutes to days.

On the other hand, formation of bimolecular quadruplexes is a second-order process and is relatively slower than unimolecular quadruplex formation. Different spectroscopic methods that probe different structural features may give different folding kinetics.

Some of these differences may result from the specific sequences or conditions [e. However, with the exception of the SPR data summarized above, the kinetic studies suggest that cation-driven quadruplex folding is a multistep process with detectable amounts of obligatory intermediates.

The intermediates are rate-limiting because relatively high-activation energies are required to produce conformational changes necessary for formation of complex topologies that may require one or more strand reversals and positioning of loops.

However, in conclusion, it is worth repeating the classical caveat of kinetics: The general principles of macromolecule stability are well known, , and for NA duplexes and to a lesser extent triplexes have been described in some detail, , , , — Although the same principles must apply to quadruplexes, the issue of the balance of forces for quadruplexes has not received much attention.

Short quadruplexes at least are significantly different from duplex and triplexes because of the nature of the H-bonding interactions, the stacking of G-quartets, and the involvement of specific ionic interactions direct inner spheres coordination as well as any nonspecific electrostatic effects of ionic interactions.

The description of the stability of any structure is best couched in terms of the free-energy contributions, as described earlier for duplex and triplex NAs. Here each component is defined as follows: The bonds term arises from bond length and angle enthalpic factors, and el refers to electronic interactions including polarization and the exchange terms involved in, for example, the hypochromic effect in the bases.

In terms of computation, the standard parsing of the free energy of stabilization of a structure is couched in simple terms that can be loosely separated into the potential energy and the entropic contributions.

For a highly cooperative two-state folding transition and see below for recent studies concerning the cooperativity and number of intermediates, the potential energy terms can be described as a molecular mechanics potential such as Equation Generally, it is assumed that all individual components are independent and harmonic 51— Until recently, the polarizability of various groups was ignored amber.

The latter depends on the details of the model being used full atom discrete model with explicit solvent versus continuum model etc. The issue of the partial charges is also complicated and they have to be carefully calculated from a relatively high-level theory, and thus for isolated nucleotides.

However, the nucleotides are aromatic systems, and as such are quite polarizeable. If one assumes local i. A fully anisotropic polarizability a tensor quantity would be considerably more involved to implement.

Current versions of mechanics programs treat polarizability in a fairly simple manner — The treatment of hydrogen bonds can be explicit or implicit, depending on the implementation. As the H-bond in biological system is largely electrostatic and van der Waal’s, these terms properly calibrated can be sufficient to account for the H-bond energy.

However, H bonds in principle have a covalent character to them, and this can be detected by for example NMR methods 84—86 , Density functional theory DFT calculations have shown how the scalar contribution depends on distance and orientation of the donor and acceptor atoms These force fields do not account for optical effects that arise from the pi stacking, including the hypochromicity and CD.

The same mechanics force field can be used both for calculated a potential energy or in principle the difference between two states, see below. As the important determinant of stability is actually the free-energy difference between the state of interest and all other possible realizable states, the entropic components also need to be understood.

The configurational entropies of the folded and unfolded states for example can be calculated from long dynamics calculations or Monte-Carlo sampling. However, the largest differences in entropy are likely to arise from the solvation and electrostatic terms, as the numbers of associated water and counterions may be very different between the compact and extended states.

As the nature of a nonfolded G-rich DNA strand is unknown, and experimentally very difficult to evaluate ensemble of conformations, variable degrees of base—base stacking that depend on temperature and salt, such calculations are presently daunting.

For this reason, modeling stability generally looks at the neighborhood of the local energy minimum of the folded state to evaluate its resistance to substantial perturbation. This is not thermodynamic stability, but may be relevant to kinetic stability.

However, calculations of free energies associated with the folding landscape have been attempted 51 , Each quadruplex is associated with the formation of two H bonds per G, or eight per quartet.

The acceptors and donors in the strand state are probably hydrogen bonding to water, so the net change in the number of H-bonds is zero, and thus the enthalpy change from intramolecular H-bond formation is relatively small However, there may be a favorable entropy change due to the release of water into the bulk solvent.

As pointed out above, there may be a small net energy gain in the folded state from the covalent character of the NH:: N H-bonds formed 85 , as well as from change in the dipole interaction energy in a region of lower dielectric permittivity compared with full exposure to water.

Generally, however, the contribution of H-bonding to the free energy of stabilization is thought to be small, , The potential energy from van der Waals interaction in a well-optimized structure is large and negative, because of the very large number the small favorable interaction energies between pairs of atoms.

As each atom is essentially close packed, then any expanded structure in vacuo will result in a large net unfavorable energy. However, in a solvent the unfolding of a NA or protein is compensated by an essentially equal number of similar van der Waals interactions with the solvent molecules, such that the net stabilization energy is rather small This is likely to be roughly linear per stack added, though the actual net favorable energy can be offset by unfavorable strain from loop lengths, which is itself structure dependent 62 , 75 , , The stacking interactions are similar to those found in other NA structures, and likely account for a substantial part of the net stabilization free energy.

The most destabilizing component in NAs at neutral pH is the very unfavorable electrostatic interactions between the oxygen atoms in the phosphodiester bonds, which bear a formal charge of —1.

This is exacerbated when a NA strand folds, and this unfavorable energy needs to be offset by all other favorable interactions, or by neutralization by counterions. In quadruplexes, the specific site binding of approximately one monovalent ion per quartet is one part of a favorable stabilization.

In quadruplexes, there are two kinds of ion binding. Such ions interact as outer sphere complexes i. The standard way of estimating number of ions released or absorbed on folding is to use thermochemistry.

The melting temperature will depend on the concentration of salt if the charge density differences between the states:. For NA unfolding, the formal charge arises for the phosphodiesters, i.

The formal charge leads to a high intrinsic electrostatic free energy, which is unfavorable. If the charge density exceeds a critical value, which depends on geometry as well as the number of charges, then ions will condense and neutralize the charge.

The critical parameter for nonspherical objects is the so-called Bjerrum length. Thus for a B-DNA polymer, about 0. In other words, a mer duplex will release The condensation model has been verified by experiment and extensive Poisson—Boltzmann calculations.

The latter have shown that short oligonucleotides deviate somewhat owing to so-called end effects the ends of a duplex have a lower charge density than those in the interior suggesting that these theoretical values would overestimate the experimental values for short oligonucleotides, More recently, Manning, has shown that even a spherical charge distribution will condense ions, depending on the actual charge density, and also on the ionic strength, albeit less than a cylinder.

Conversely, if a folded, intramolecular fold such as a quadruplex were to condense less ions than the unfolded state, then the salt-dependent stability would be the reverse of the duplex-strand transition, i.

Unfortunately, there seem to be no Poisson—Boltzmann calculations carried out on small, well-defined quadruplexes, for which one might imagine that the specific of topology and thus charge density would be important.

The uptake per mole of formal negative charge varied from 0. However, not all of the phosphates are involved in the G-quartet stacks i. If the uptake of ions is normalized, rather arbitrarily, to the number of nonloop phosphates, the uptake is much larger, i.

These are still low numbers when one takes into account also that these structures are binding 2—3 ions specifically, i. Again, the binding of two to three specific ions in the G-quartets would itself account for 0.

Although the discrepancies among the various sequences and methodologies are substantial, the number of ions taken up per quadruplex formed is comparable to the expected number of specific ions bound, suggesting that the ionic strength effects may be quite small compared with the more familiar duplex folding.

Given the uncertainties regarding ion condensation, it is unclear whether these numbers are merely fortuitous. Nucleotides exposed to solvent unfolded states are hydrated.

However, approximately four water molecules per nucleotide is released on unfolding of DNA duplexes, It is possible to determine the amount of water released or taken up during a transition by using osmolytes to affect the water activity, provided they can be shown to be true osmolytes and do not interact directly with either state of the solute of interest, Such studies have been carried out for duplex and triplex DNA It is also possible to combine electrostatics and hydration simultaneously by varying the concentration of salt, which alters water activity.

In DNA duplex unfolding, the stabilizing effect of increasing ionic strength is opposed by the decrease in water activity as the duplex is more hydrated than the strands, e.

The first term on the right-hand side of Equation 13 is the nonideality effect of salt on the DNA, the second term represents the decrease in water interaction due to replacement by salt, the third is the nonideality term from salt—water interactions and the final term is the electrostatic salt—salt nonideality effect.

The nonideality and cross terms in Equation 14 become especially important at higher salt concentrations, such as approaching the 1 M standard state often used in NAs thermodynamics where the polyelectrolyte contribution is zero, This is opposite to DNA duplex folding, which is associated with an uptake of water, Small molecule osmolytes thus stabilize the quadruplexes.

Further, other cosolvents such as primary alcohols that not only decrease the water activity, but also the bulk dielectric constant, were shown to increase quadruplex stability, which is opposite to the well-known effect of alcohols on DNA duplex solubility and thermodynamic stability.

This behavior was interpreted in terms of favorable electrostatics for folding. In a formal sense, the ion binding is described by a simple thermodynamic square as shown in Scheme Here if m, n are greater than 1, it is assumed that the cooperativity of binding is very high cf.

The temperature dependence of folding, as in UV melting for example, would be This becomes a linear function of when K 4 is large compared with, and K 2 is small compared with. The slope approaches n, the number of ions bound in the folded state.

In an optical titration, the observed signal depends on the specific absorption coefficient. The assumption that metal binding does not in itself affect the absorption coefficients of the nucleotides is for illustrative purposes only and represents the simplest case.

Equation 16 is a Hill equation and will show positive binding cooperativity of L at a fixed temperature. In most cases, the values of K 1 would be large, so the apparent dissociation constant for e.

Under such conditions, the Hill coefficient n is an indicator of the number of ions bound during folding These affinities are apparent as they are accompanied by folding, as in Scheme 2. As the specific ion binding to the unfolded state is also likely to be small, the stabilization due to specific ion binding should be substantial i.

This corresponds to 7. As oxygen ligands are replaced by oxygen ligands, one can envision that the enthalpy change of pure ion binding is relatively small. This further implies that under appropriate circumstances, the population of the unfolded state in the absence of cation could be significant.

This in general accord with the kinetics of formation of the human telomere quadruplex initiated by sodium or potassium in which cation binding is highly cooperative, both under equilibrium conditions and in the early phase of the approach to equilibrium.

Nevertheless, this analysis is highly simplified, and really reflects our current lack of understanding of the energetics of quadruplex formation and stability. Hud and coworkers 31 have recently summarized the literature on ion binding to G-quadruplexes.

The difference in stability between sodium and potassium forms is well documented, though there is a tendency for these ions stabilized different structures, in which the coordination stereochemistry of the ions also differ cf.

Experimentally, the difference in free energy between potassium and sodium binding is of the order 1. This is in agreement with calculations that indicate essentially free mobility of small ions e.

An actual parsing of the component energies has yet to be achieved. In Quadruplex topologies and structures section, we alluded to the restrictions imposed by loop length on possible topologies and by implication the contribution to the overall stability of different quadruplex structures.

Although we have argued so far that bases stacking and specific ion binding may be the dominant stabilizing interactions, the length and sequence of these loops is important. Similarly, replacing nucleotide with nonnucleotide linkers had substantial effects on both stability and folding kinetics Loop length clearly influences the energetics of quadruplexes, because changing the length from 1 nt to 2 or 3 is associated with a change on overall fold In general, one would expect the stability to increase with the number of stacks.

Similarly, in the series GnT2 4 , the free-energy change was also linear in n, apart from one anomaly, with a slope of 1. Interestingly, the apparent number of potassium ions taken up on folding barely increased with n from 2.

The presence of the T2 loops was suggested to allow for different topologies antiparallel rather than parallel Nonspecific ion dependencies might also account for some of the anomalous estimates of the number of ions bound, as may the annealing history of the nucleotides e.

In any event, this all points to the complexity of dealing with quadruplex structures, and the need for simple but rigorous methods to establish what structures are present and the mole fractions under the experimental conditions when multiple conformations are present.

Because thermodynamics refers to changes in state functions, all thermodynamic considerations must include nature of the unfolded state. Even very high quality energy calculations on a particular structure imply little about net thermodynamic stability.

The rather dense purine sequences of G-tracts suggest that the single stranded state at normal temperature and salt conditions is unlikely to be remotely like an extended strand or a random coil; significant base stacking of nearest neighbors is to be expected.

Telomeric DNA can be prevented from forming quadruplex structures by ensuring that there are no high Z cations present. A macrocation such as the tetramethylammonium TMA ion will also prevent quadruplex formation better than Li, because the ion is simply too large to fit into the cavity of the quartet.

However, its CD spectrum has a classical conservative exciton coupling shape, indicative of significant base stacking, but not consistent with G-quadruplex folded structures R. Thus, the strand state is by no means a random coil.

In principle, FRET could be used to assess the distribution of end-to-end distances in the unfolded state Such FRET experiments in TMA might provide useful information about the distribution of size and shape in the unfolded form, and thus the degree of stacking in the strand state.

Given the preceding brief overview, we now discuss the factors that determine formation of known structures, and what can actually form, in terms of kinetic versus thermodynamic control, and how these might be modulated by cellular environments.

NA folding is associated with altered hydration. The effect of water activity can be estimated using osmolytes to influence the water activity, and the data can be analyzed in terms of preferential hydration as described by Timasheff — and by Parsegian, The dehydration would stabilize quadruplexes compared with duplexes, which are more hydrated than the single strand and thus the quadruplex state.

This macromolecular matrix in itself disregarding for the moment any specific or nonspecific direct interactions can influence folding energy and kinetics as described by Minton, based on considerations of the coefficients of the virial expansion of state.

Both theoretically and experimentally, it is shown that the presence of noninteracting matter will favor the more compact state, i. Note that this would synergize the effects of water activity on hydration.

Sugimoto, have used crowding conditions in vitro to determine the influence on conformation and stability. They found that the crowding conditions caused a change in the structure of the quadruplex, owing to different excluded volumes cf.

Interestingly, as the crowding also decreases the water activity, it was argued that crowding conditions stabilized the parallel G-quadruplex structure because it releases water on formation Separating the contributions from crowding, solvation and electrostatics is complex, as they are not linearly independent parameters [cf.

A degree of separation may be achieved by using a range of small molecule osmolytes to probe water activity effects, and large, truly physically excluded noninteracting polymers for crowding effects in comparison with the small molecules, In vivo this is much more complex, because the proteins present in the nucleus are clearly not neutral particles, but rather a collection of architectural, structure and sequence selective molecules.

The folding of a single-strand G-rich overhang such as the one that exists in telomeres is a very different proposition from the formation of a G-quadruplex in an internal position, such as identified in numerous promoters 60 , This area of research has been recently reviewed In the former case, the folding is free to occur from an unconstrained end, and does so on a potentially stabilizing nucleus of double-stranded B-like DNA In contrast, from the point of view of energetics, the internal G-quadruplexes require the separation of the DNA strands to form a loop very unfavorable followed by the G-rich strand forming a quartet while being tethered at both ends, leaving the complementary strand as a loop, or as an i-motif as shown in Figure 9.

Based on estimates of the energies of formation of these structures, , , it is possible to calculate the cost of formation of such a structure, as follows. The T m depends on the fraction GC and the ionic strength as The values of T m for three different GC contents and three ionic strength values are given in Table 3.

The ionic strength covers the range used experimentally and the average intracellular monovalent ion content. Note that the duplexes are stabilized by salt less than the quadruplexes. The van’t Hoff enthalpy estimates from ref.

Extrapolation to a common reference temperature can also lead to error. To some extent, this energy cost might be offset if the complementary strand were to form a stable structure, such as the i-motif — However, this structure is at best marginally stable at physiological pH and temperature though the actual state of protonation of the cytosines in the nucleus is far from clear.

The reason for this is that the i-motif involves H-bonding between cytosines, on which one of the pair must be protonated on N3. Thus, these structures are stable only at low pH This does not include the unwinding stress and junction with neighboring duplex, although it is possible to build models, as yet unrefined, that simultaneously incorporate a G-quadruplex and an intramolecular i-motif on the complementary strand 65 , Figure 9.

Experimentally, the duplex wins over the quadruplex, , , as expected on thermodynamic grounds see above. An alternative is that a quadruplex becomes stabilized by a protein or other ligand, or that the single-stranded complement is similarly stabilized by a binding partner, such as a DNA.

RNA hybrid as in Escherichia coli 8 , which is typically a rather short hybrid duplex that is important in transcription initiation, or a ss binding protein. Another alternative is that supercoiling stress generated by transcription might supply this energy even transiently, though one might expect the less AT-rich regions to form a bubble in preference to a GC-rich sequence, unless again there are additional proteins that could stabilize on the other strand.

It is clear that such a structure will not spontaneously form without some help. It has been suggested that supercoiling such as during transcription can propagate the necessary stress back toward the promoter to cause unwinding of sequences, and thus permit the formation of the G-quadruplexes It is unclear why AT-rich sequences would not melt in preference to the GC-rich sequences, given the very large difference in stability.

Furthermore the applied torsional stress would surely change as the transcription proceeds, so that at some point the G-quadruplex would revert to the duplex state. However, as the kinetics of reversion are typically very slow, this might not be possible unless other proteins were to accelerate the kinetics.

Nevertheless, the implication was also that the system operates far from equilibrium, implying that an understanding of the kinetics is extremely important. Indeed, it has been proposed that specific DNA-binding proteins bind to the single strand region in the torsionally stressed Myc promoter Risitano showed that in fact the ds moiety of the human telomeres is preferentially double stranded at physiological pH, but an increase the number of G-stacks favored the quadruplex over the duplex such as for the c-myc G-rich sequence, even in the presence of a large excess of the complementary C-rich strand The excess C-rich strand 5- to fold is thus in very large excess of the values of K 1 K 2 , and this should easily outcompete the quadruplex for all sequences studied, in agreement with, , For an internal loop, the penalty will be higher than for a free duplex Major arguments in favor of G-quadruplex formation in vivo include the ready ability of the G-rich sequences widely found in genomic DNA, as single strands spontaneously form a stable quadruplex structures in solution.

Furthermore, the prevalence of such G-potential sequences that far exceeds chance is argued to be preserved for a functional purpose. The latter argument has considerable weight but not specifically for the formation of any particular structure.

As discussed earlier, thermodynamic arguments favor the telomere as the most likely place to find G-quadruplexes in vivo. Such a structure would however protect the end of the chromosome from unwanted dimerization which would interfere with replication and with subsequent accurate chromosome segregation during mitosis, i.

It is experimentally very difficult to demonstrate the presence on any specific DNA structure in vivo and even harder to prove its absence! Maizels 12 and Bryan 14 have recently summarized the evidence for the existence of quadruplex telomeric G4 DNA in vivo [see Kipling, pp.

An overview of the differences in complexity of telomere replication among various organisms is given by Gilson and Geli The best evidence exists for ciliates such as Oxytricha and Stylonychia which have a very different kind of chromatin than mammals and a very large number of telomeres.

The fundamental paper by Schaffitzel et al. These two antibodies showed no significant affinity for any other kinds of DNA or RNA, or other polyanions although no proteins were tested.

Only the latter showed a positive result in the indirect immunostaining technique on isolated nuclei from Stylonychia lemnae, and then only in the vegetative state i. Indeed, the multiple chromosomes from the vegetative state tend to aggregate end-to-end in this organism, via the intermolecular G-quadruplex, and this may explain why the in vivo chemical modification experiments failed to detect quadruplex in these organisms, For subsequent replication, this complex must be resolved.

It is worth noting that the observed rate constant for the approach to equilibrium as opposed to the unidirectional rate constants and thus the half-life for formation and dissociation of a structure are the same.

Consider the simple intramolecular folding mechanism:. If one were to start the reaction with adding ion to unfolded nucleotide, the formation of Q will proceed exponentially toward equilibrium, according to:.

In contrast, the spontaneous unfolding from Q will occur with the same rate constant k obs provided that there is nothing to mop up the resulting strand or ions. Only the initial rates differ in the two directions.

Anything that binds Q will slow down the rate of approach to equilibrium, and similarly anything that sequester U will do so too, as the rates depend on the concentration of U and Q.

A major conclusion, ultimately based on known in vitro properties of G-rich oligonucleotides, is that if quadruplexes are formed, they need to be resolved by proteins, and cells appear to devote considerable effort to prevention of G-quadruplex formation, because they are too stable for replication or transcription.

Of the 21 proteins listed as being able to bind to quadruplex DNAs in vitro, only one quadruplex was a fold-back structure Many other proteins can be shown in vitro to have affinity for such structures, but their functional significance is unclear.

Recently, it has been argued that only the extreme end of vertebrate telomeres form quadruplexes, and a protein proposed to be needed for human telomere unfolding has been characterized Much of the evidence for quadruplex formation in mammalian DNA in vivo is generally circumstantial and to date no high selectivity antibodies for human quadruplex structures are available.

Quadruplex binding ligands typically have rather modest affinity and selectivity even for the small numbers of structures tested, and do not always bind in the manner expected or designed, , New developments in quadruplex-binding ligands show substantially higher affinity and selectivity for the human telomere sequence, and these might be useful for such experiments, with the caveat that the ratio of nonquadruplex DNA to potential quadruplex DNA is very large, and that selectivity also needs to be assessed against protein binding and a wide variety of alternative DNA structures.

The biology of telomeres was summarized in a recent review regarding their need for maintenance, and what happens when the length is not maintained This is called the t-loop mechanism — Although the length of the telomeres is much greater than the persistence length of DNA at physiological salt concentrations, opening of an internal loop within the duplex and forming in essence an intermolecular duplex is, as explained above, energetically unfavorable.

In fact, it was observed that in the absence of a stabilizing factor, the efficiency of t-loop formation was very low; only substantial formation of a loop structure was observed in the presence of the telomere specific protein TRF2 Indeed, the loop model does not formally require strand invasion, only that there is some interaction stabilized by for example protein.

Indeed, it has recently been argued that just the single strand-overhangs form a t-loop stabilized by what amounts to a dimeric G-quadruplex However, this is not in agreement with original models based on large loops that imply circularization of the lengthy double-stranded portion of the telomere.

Although the binding energy for a loop of this size would be small if the DNA were naked, the entropic cost could be quite substantial, as the probability of finding a small number of configurations in which the end is close to a specific part of the telomere is small compared with all the other possible configurations We note that the chromatin rearrangement implies connection of the physical problem to metabolism via ATP hydrolysis and acetylation events.

This also begs the question as to the remarkable length variation of telomeres, even in mammals. It is clear from the foregoing that despite the extensive efforts expended to date, we still have a very limited view of what determines the folding and relative stability of different quadruplex structures, and how fast they form.

Even worse, for the most part the relationship to in vivo formation is even murkier. The latter is a consequence of at least two factors. First is that in vitro experiments concentrate on simple, relatively well-defined systems under conditions that typically do not approximate those found in vivo, as described in part in Discussion section.

Furthermore, it turns out that even the short intramolecular complexes are surprisingly complex, and detailed analysis is hampered by the presence of multiple conformations and slow folding kinetics.

Nevertheless, the basic principles of the energetics of G-quadruplex formation do need to be considered in any biological model, as well as the question as to where the energy comes from.

Thus, the thermodynamics and kinetics of small oligonucleotides have value in that it is possible to examine them in great detail under a wide variety of well-controlled conditions. This allows one to sample the possible range of behaviors within a realistic range energies and timescales, which ultimately must be relevant to the biological conditions.

It appears that naked G-potential DNA alone has physical properties that are not commensurate with some of the proposed biological functions and thus requires protein binding to provide the energy to manipulate these properties.

It is further notable that some of the proposed functions of such DNA, such as found in promoters, may need to be coupled to unwinding events, and operate far from equilibrium. Under these conditions an understanding of kinetics is essential.

We have argued that the specific ion binding and general electrostatics are a critical component for the energetics, and folding rates of G-quadruplexes, that distinguish them from most other DNA or RNA folds.

However, there are disturbing variations in basic thermodynamic properties that need to be addressed to establish whether these variations arise from experimental artifacts or there are real differences that are sensitive to conditions and small variation in sequence.

To accomplish this goal, we believe that an agreed upon and reliable set of rapid and inexpensive techniques is needed to determine the topology of any quadruplex, and assess its purity.

This is likely to come from a combination high-resolution spectroscopic and hydrodynamic techniques, such as NMR and AUC. First, methods are needed for rapid assessment of the topological structure s that is are present and whether the state is unique and thus can be manipulated by variation of conditions until it is.

This is a prerequisite for subsequent structural and functional analysis, purely as an analytical quality control. Second, it would make it possible to prepare a standardized set of oligomers of known structures to determine whether even simpler methods that exist can make useful distinctions among possible structures database approach.

To this end, we propose that a consortium be established to address major unresolved questions, or at least enlighten the present authors who confess having more questions than answers.

Some of these are listed below. Finally, there is a substantial literature on targeting G-quadruplexes for possible anticancer drug development 18 , 60 , which is beyond the scope of this review.

However, the physical principles apply equally to this area. This work was funded in part by NIH grant No. Funding to pay the open access charge for this article has been waived by NAR.

We thank Paula J. Bates and a reviewer for a critical reading of the article and valuable comments. Your ideas and opinions can help provide a better service for the life sciences research community.

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For the fiscal year, the Committee received the advice of the compensation consultant about the size of the restricted stock and stock option grants for senior officers and generally followed that advice.

Foss is still employed by the Company on that date. During the fiscal year, an additional long-term incentive program was instituted for senior management using restricted stock under the Stock Incentive Plan.

In the fiscal year, the Company ranked in the third quartile in its peer group based on total shareholder return. Similar to the retirement benefit restoration plan, the deferred compensation plan and the k Savings Plan, together allow executives affected by the Internal Revenue Code limits the opportunity to make and receive contributions at approximately the same percentage of compensation as are made and received by other employees.

One of the Named Executive Officers participated in this plan during the fiscal year. The Committee believes that Mr. McCollough has reduced expenses and led necessary changes in the areas of store design, merchandising and marketing.

For fiscal, the Committee maintained Mr. McCollough did not receive a performance bonus for the fiscal year. The Committee believes that the structure of incentives to Mr. McCollough is appropriate for Mr.

The Committee views his fiscal compensation as appropriate when compared with the results of the Company, given the changing market conditions. During fiscal, directors who were not employees of the Company received a combination of equity-based and cash compensation.

Non-employee directors are not permitted to sell or transfer the shares underlying the restricted stock units granted until the shares are fully vested and the non-employee director either retires or ceases to be a director of the Company.

If a non-employee director attends less than 75 percent of the aggregate Board and committee meetings during a year, he or she forfeits 50 percent of their cash compensation earned for the year.

Equity-based awards are generally made on the date of the annual meeting. Hardymon, Kane, Salovaara, Ms. Feigin deferred this stock retainer grant for fiscal As a result, Allen B.

King elected to defer this stock retainer grant for fiscal Directors who are employees of the Company receive no compensation for service as members of the Board or Board committees.

Officers, directors and greater than 10 percent shareholders are required by regulation to furnish the Company with copies of all Forms 3, 4 and 5 they file. Amendments to the Purchase Plan.

Purpose and Principal Features of the Purchase Plan. The Board has adopted, and recommends that the shareholders approve, the amendment and restatement of the Purchase Plan. The Purchase Plan was originally adopted by the Board in Although it has been amended over the past 20 years to change the mechanics of its operation and to make additional shares available for purchase, the basic purpose of the Purchase Plan has remained the same.

The Purchase Plan is intended to provide a means through which the Company can encourage and assist employees of the Company and its subsidiaries to acquire stock ownership interests in the Company.

The Purchase Plan assists the Company and its subsidiaries in. Shares Available for Purchase. Based on the number of shares outstanding at December 31, , the Senior Officers could have increased the number of shares reserved under the Purchase Plan by up to 2,, shares for calendar year The Committee has the full power, discretion and authority to interpret and administer the Purchase Plan and the rights granted under it.

The Committee has also appointed a Plan Administrator to assist the Committee in carrying out its responsibilities. The Plan Administrator is responsible for the general administration of the Purchase Plan.

Participation in the Plan. Compensation for purposes of the Purchase Plan generally includes cash compensation and commissions, including overtime and bonuses. Employees may also reinvest dividends received on shares held in their Purchase Plan accounts in common stock, although there is no Company match on the purchase funded through dividend reinvestment.

The amount of the Company match is set by the Board and may be changed by the Board at any time. A Participant whose participation is terminated may leave the shares in his or her Purchase Plan account, sell the shares or request a certificate for the shares.

Rights under the Purchase Plan are not transferable by a participating employee. Duration, Amendment and Termination. Federal Income Tax Information. Under the Internal Revenue Code of, as amended, this income constitutes wages, subject to withholding for income and payroll taxes.

Dividends are taxable income even if the employee reinvests them in common stock and does not keep them as cash. There are no federal income tax consequences to the Company by reason of the purchase of shares under the Purchase Plan.

The Company will be entitled to a business expense deduction for the Company matching contribution, at the time and in the amount that the related income is reported for the participating employee.

The Purchase Plan, as amended and restated, will be approved if the votes cast in favor of approval exceed the votes cast against approval. The principal reason for the amendment relating to the number of shares issuable is to allow the Company to continue to make equity awards under the Plan.

There are presently approximately 24, shares of common stock available for granting under the Plan. Principal Features of the Plan. The Board administers the Plan and has the complete discretion to determine when to grant incentive awards; which eligible non-employee directors will receive incentive awards; whether the award will be an option, a stock appreciation right, restricted stock, restricted stock units or stock; and the number of shares to be allocated to each incentive award.

The Board may impose conditions on the exercise of options and stock appreciation rights and on the transfer of restricted stock received under the Plan and may impose such other restrictions and requirements as it may deem appropriate.

The Plan is intended to conform to the provisions of Rule 16b-3 of the Securities Exchange Act of, as amended. Options may be exercised only at such times as specified by the Board.

If the option provides, an optionee exercising an option may pay the purchase price in cash, by delivering shares of common stock, or by delivering an exercise notice together with irrevocable instructions to a broker to promptly deliver to the Company the amount of sale or loan proceeds from the option shares to pay the exercise price.

The Board may award stock appreciation rights under the Plan. When the stock appreciation right is exercisable, the holder may surrender to the Company all or a portion of the unexercised stock appreciation right and receive in exchange an amount equal to the difference between i the fair market value on the date of exercise and ii the fair market value on the date the stock appreciation right was awarded.

Restricted stock units may be granted on the terms and conditions set by the Board with the same limits as for restricted stock. In the case of restricted stock units, no shares are issued at the time of grant.

Rather, upon the lapse of all restrictions, a restricted stock unit entitles a participant to receive shares of common stock or a cash amount equal to the fair market value of a share of common stock on the date the restrictions lapse.

This summary of the principal features of the Plan is qualified by reference to the actual provisions of the Plan, a copy of which is available to any shareholder or director upon written request to the Company.

Options and stock appreciation rights may be transferable by a participant and exercisable by a person other than a participant, but only to the extent specifically provided in the terms of the award.

Federal Income Tax Consequences. A non-employee director does not incur federal income tax when granted a nonstatutory stock option, a stock appreciation right, restricted stock or restricted stock unit.

In general, a non-employee director who has received shares of restricted stock or restricted stock units will include in gross income as compensation income an amount equal to the fair market value of the shares of restricted stock or restricted stock units at the time the restrictions lapse or are removed.

Such amount will be included in income in the tax year in which such event occurs. A non-employee director will incur federal income tax when he or she is awarded a stock grant.

In general, a participating director who has received a stock grant will include in gross income as compensation income an amount equal to the fair market value of the shares at the time of grant.

This summary of federal income tax consequences of incentive awards granted under the Plan does not purport to be complete. State, local and foreign income taxes also may be applicable to the transactions described above.

Representatives of KPMG LLP will be present at the Annual Meeting, will have the opportunity to make a statement if they so desire and will be available to respond to appropriate questions.

Independent Auditor Fees and Pre-approval Policies. Audit Fees include fees billed for the audit of the consolidated financial statements, quarterly reviews of unaudited financial statements, accounting advice and services related to registration statements filed with the SEC.

Audit Related Fees include fees billed for audits of employee benefit plans, Sarbanes-Oxley documentation assistance, attestation services related to securitization activities and due diligence assistance on a proposed acquisition.

All other services must be approved by the Audit Committee. At this time, the Company does not know of any other business that will be presented to the meeting. If any other business properly comes before the meeting, your proxy may be voted by the persons named in it in such manner as they deem proper.

In addition, Section 1. The notice must be received 1 on or after February 1st and before March 1st of the year in which the meeting will be held, if clause 2 is not applicable, or 2 not less than 90 days before the date of the meeting if the date for that meeting prescribed in the bylaws has been changed by more than 30 days.

A proposal that any shareholder desires to have included in the proxy statement for the annual meeting of shareholders must be received by the Company no later than January 8, The purpose of the committee will be to: In its capacity as a committee of the Board of Directors, be directly responsible for the appointment, compensation, retention and oversight of the work of the independent auditor.

Consider, in consultation with the independent auditor, the internal audit directors and the chief financial officer, the audit scope and plan of the independent auditor and the internal auditors.

Role of the Audit Committee. This charter assigns oversight responsibilities to the audit committee. The independent auditor is responsible for performing an independent audit of the consolidated financial statements in accordance with generally accepted auditing standards.

The members of the audit committee are not acting as experts in accounting or auditing and rely without independent verification on the information provided to them and on the representations made by management and the independent auditor.

The Circuit City Stores, Inc. For the purposes of the Plan the following terms have the stated definitions. Additional terms are defined in the sections below. Amount of Stock Subject to the Plan.

The total number of shares of Common Stock which may be purchased under the Plan shall be 15,,, subject to adjustment as provided in Section Such shares may be newly issued shares that have been authorized but not yet issued or may be shares purchased for Participating Employees on the open market.

All present and future Employees of the Circuit City Companies who have been employed by a Circuit City Company for at least one year are eligible to participate in the Plan, except: If an Employee has one year of service but is excluded from participation in the Plan due to the requirements set forth in i or ii in the preceding paragraph, the Employee will be eligible to participate in the Plan on the first Enrollment Date after he or she is no longer excluded because of such requirements.

Administration of the Plan. The Plan shall be administered by the Committee. Any construction, interpretation, or application of the Plan by the Committee shall be final, conclusive and binding.

An Employee seeking to participate in the Plan must deliver an Enrollment Form to the Benefits Department so that it is received sufficiently prior to the Enrollment Date to allow processing by the Benefits Department.

The Plan Administrator may establish a submission deadline for Enrollment Forms. Payroll Deductions and Limitations. The Plan Administrator shall have the power to change these percentage limitations.

Changes in Payroll Deductions. A Participating Employee may change the percentage of his or her payroll deductions, subject to the minimum, maximum and allowed increments set forth in Section 6.

To accomplish this, the Participating Employee must submit to the Benefits Department a new Enrollment Form stating the new deduction percentage. The change will be effective as of the first of the next month if the Enrollment Form is received sufficiently prior to the first of the month to allow processing by the Benefits Department.

Deadlines for submission of Enrollment Forms for the purpose of changing payroll deductions may be established by the Plan Administrator. A Participating Employee may also elect to stop making contributions in the manner described in Section Here we report a simple chromatographic technique that separates the individual folds without need for sequence modification.

However, NMR and chromatography showed that this oligonucleotide produces at least eight monomeric quadruplex species that interconvert very slowly at room temperature.

We have used a combination of spectroscopic, hydrodynamic and thermodynamic techniques to evaluate the physicochemical properties of the mixture and the individual species.

These species have almost identical thermodynamic, hydrodynamic and electrophoretic properties, but significantly different NMR and circular dichroism CD spectra, as well as kinetic stability.

These results demonstrate that simple standard low-resolution techniques cannot always be used for quadruplex fold determination or quality control purposes, and that simple thermodynamic analysis does not directly provide interpretable thermodynamic parameters.

In the presence of appropriate cations, nucleic acid sequences of the general kind GGGN n spontaneously fold into stable three-dimensional structures. These are characterized by the stacking of two or more successive planes of four guanine residues arranged in a square planar array via Hoogsteen hydrogen bonding a G-quartet.

Such sequences are found naturally in the telomeres of chromatin in all species so far examined 2—6 , as well as in internal sites of DNA, especially in promoter regions 7—9.

The prevalence of these latter sequences is very high 7. In general the stability of quadruplex structures is greater in the presence of potassium rather than in sodium solutions 10 , 11 , and it has been suggested that G-rich sequences may occur in vivo under appropriate circumstances, either as regulatory elements 2 , 12 , 13 , or for as yet unknown reasons.

There are several G-quadruplex aptamers that have therapeutic uses, and are presently undergoing phase I and phase II clinical trials 16 , A notable example is AS, which is now in phase II clinical trials.

Quadruplexes have also been shown to have antiviral activity and have been demonstrated to be effective against HIV-1 in vitro 18 , Because of their ability to recognize both nucleic acids and proteins with a high degree of specificity combined with their stability and nuclease resistance, quadruplexes are rapidly becoming attractive agents for development of novel therapeutics and preclinical studies are underway for several other quadruplex-based therapeutics 20— It has been shown that G-quadruplex-forming sequences are remarkably polymorphic.

A single sequence may fold into several structures depending on the counterion residing in its core, the details of the folding conditions, and the environment. Of the 26 possible looping topologies and 8 possible tetrad arrangements available based on the glycosyl bond angles orientations, there are over possible unimolecular conformations for the simplest quadruplex The formation of multistranded structures further increases the possible conformational space.

Furthermore, the conformational polymorphism observed under solution conditions does not adequately reflect the conditions in vivo, including crowding, specific protein binding and the fact that such sequences are closed at one telomeres or both ends internal DNA sites such as promoter regions 29 , Polymorphism seems to be a common feature of G-rich sequences 1 , Whether this occurs in biological systems or only in vitro, it is important to understand the rules that determine the populations of the various structures under given conditions, especially for biotechnology applications.

Many quadruplex systems cannot be described structurally due to high complexity resulting from the simultaneous presence of multiple species. Ideally one would simply separate the various species present in solution in order to analyze them individually.

Unfortunately, for G-quadruplexes it is difficult to apply standard separation techniques because, as we show here, different folds of the same sequence often have nearly identical physical properties.

This may contribute to the inconsistencies in the reported structural and biophysical data for differently modified systems derived from a single sequence. There are several theoretically possible monomeric, dimeric or tetrameric G-quadruplex topologies for this sequence as shown in Figure 1.

In practice, it folds into at least eight different monomeric quadruplex structures under the same experimental conditions. Furthermore, a different species is produced by rapid cooling, indicating significant differences between kinetic and thermodynamic stability.

We will show that many of these species can be resolved by chromatography. We have characterized individual conformers by several biophysical techniques, and compared them both with one another and with the unresolved mixture.

We have demonstrated that each conformation has very similar hydrodynamic and electrophoretic properties, and similar thermodynamic stability, yet can be resolved by chromatography into species that have very distinct nuclear magnetic resonance NMR.

With one exception, the circular dichroism CD spectra could not discriminate among the different species. In the absence of sufficient resolution or incomplete theory for most commonly used biophysical techniques 36 , and because the G-rich oligonucleotides commonly adopt multiple conformations, analysis of the quadruplex system should begin from rigorous characterization by high information content, high resolution techniques such as NMR, before detailed analyses of other biophysical properties are initiated, as we have previously suggested The oligomer was shown to be a single chemical species by electrospray mass spectrometry, high performance liquid chromatography HPLC and electrophoresis.

The guanine in the linker region G14 of the sequence was not included in the series. The DNA samples were then refrigerated until use. For calibration purposes the column was run at a flow rate of 0.

Elution was monitored at A For DNA separation, the flow rate was altered to 0. Gels were stained with ethidium bromide Sigma-Aldrich, St. Samples of individual fractions were collected from the FPLC, combined, and concentrated using microcon YM-3 Millipore centrifugal concentrators.

CD spectra of samples having an A of 0. The spectral band-width was set to 2 nm and data pitch to 0. Spectra were recorded with a response time of 2 s. Three spectra were averaged, and the buffer baseline was subtracted.

The T m was estimated from the first derivative of the melting profile at nm. The baselines outside of the melting transition were assumed to be linearly dependent on temperature according to:. These equations refer to single melting and unfolding transitions, which are the same if the system approaches equilibrium at each temperature.

Non-linear regression analyses were carried out using Kaleidagraph Synergy Software. Sedimentation velocity profiles were recorded of unfractionated and fractionated AS using a Beckman Optima XLA ultracentrifuge equipped with absorbance optical system and An Ti rotor.

All samples were dissolved in the FPLC buffer to working concentrations resulting in 0. Sedimentation coefficients showed little dependence on concentration under these conditions. Equilibrium centrifugation provided an independent estimate of M.

A value of 0. Under these conditions, all of the quadruplex converts to duplex 30 , and with a rate constant that largely represents the unfolding rate constant of the quadruplexes under the same conditions Both samples also had the same relative electrophoretic mobility, and appeared as a single band, as shown in Supplementary Figure S1.

Furthermore, the melting profile of the annealed oligonucleotide was very similar to that of the cooling profile, suggesting a reversible process at this heating and cooling rate. The sedimentation velocity profile, c S Supplementary Figure S2 of the mixture shows two species with sedimentation coefficients of 2.

The larger mass corresponds to a dimer. We conclude therefore that the oligonucleotide forms a mixture of stable monomeric and dimeric G-quadruplexes. The frictional ratio of the monomers Equation 3 is close to unity, and given that nucleic acids are typically well hydrated 40 , 44 , 45 , this suggests that the monomeric species is highly compact, and behaves hydrodynamically as a nearly spherical object.

Although the biophysical data are consistent with the presence of a monomeric quadruplex species and a certain amount of dimer, these low-resolution techniques do not unequivocally discriminate the several possible folding topologies Figure 1 , and thus experimentally generate averages.

To reveal the real sample content requires high-resolution structural techniques, such as NMR. The frequency range of the imino proton resonances Figure 3 shows a chromatogram of the slowly annealed AS The first three fractions account for two-third of the total.

For comparison, a 10 kDa protein elutes at the same volume as fraction 2. However, the CD and sedimentation velocity profiles show that not only are these species quadruplexes, but also that they are monomers having a Stokes radius and mass the same as that of the other fractions, and thus are not breakdown products Table 1.

Remarkably, all of the fractions 1—5 show a very similar mobility to that of the parent mixture. As the fractions all have the same frictional ratio Table 1 this implies that the effective charge distributions are also similar.

Fraction 1 is a mixture of monomer and dimer species, and appears as a poorly resolved doublet by gel electrophoresis, with a slower band migrating at ca. Thus, electrophoresis does not necessarily resolve multiple isomeric species; a single band is not conclusive evidence of a pure component.

These spectra are nearly isodichroic at nm. These bands correspond to Cotton effects in the absorption spectra, which have a maximum at nm, and a shoulder at nm not shown. These are typical of G-quadruplex CD spectra 36 , As we will show, this species has a substantially different NMR spectrum from that of the other fractions.

The annealing curves for fractions 1—5 are extremely similar as expected Figure 4. Fraction 1 shows a broad overlap of resonances in the NMR spectrum Figure 5 , many in common with those of the mixture.

This species is known to be a mixture of a monomeric and dimeric quadruplex forms Table 1 and presented AUC data. Fractions 3 to 5 are all monomeric, and the imino proton resonances are sharp and relatively well resolved.

The reset to kx – dapic how 922v full download

We have used a combination of spectroscopic, hydrodynamic and thermodynamic techniques to evaluate the physicochemical properties of the mixture and the individual species. These species have almost identical thermodynamic, hydrodynamic and electrophoretic properties, but significantly different NMR and circular dichroism CD spectra, as well as kinetic stability.

These results demonstrate that simple standard low-resolution techniques cannot always be used for quadruplex fold determination or quality control purposes, and that simple thermodynamic analysis does not directly provide interpretable thermodynamic parameters.

In the presence of appropriate cations, nucleic acid sequences of the general kind GGGN n spontaneously fold into stable three-dimensional structures. These are characterized by the stacking of two or more successive planes of four guanine residues arranged in a square planar array via Hoogsteen hydrogen bonding a G-quartet.

Such sequences are found naturally in the telomeres of chromatin in all species so far examined 2—6 , as well as in internal sites of DNA, especially in promoter regions 7—9.

The prevalence of these latter sequences is very high 7. In general the stability of quadruplex structures is greater in the presence of potassium rather than in sodium solutions 10 , 11 , and it has been suggested that G-rich sequences may occur in vivo under appropriate circumstances, either as regulatory elements 2 , 12 , 13 , or for as yet unknown reasons.

There are several G-quadruplex aptamers that have therapeutic uses, and are presently undergoing phase I and phase II clinical trials 16 , A notable example is AS, which is now in phase II clinical trials.

Quadruplexes have also been shown to have antiviral activity and have been demonstrated to be effective against HIV-1 in vitro 18 , Because of their ability to recognize both nucleic acids and proteins with a high degree of specificity combined with their stability and nuclease resistance, quadruplexes are rapidly becoming attractive agents for development of novel therapeutics and preclinical studies are underway for several other quadruplex-based therapeutics 20— It has been shown that G-quadruplex-forming sequences are remarkably polymorphic.

A single sequence may fold into several structures depending on the counterion residing in its core, the details of the folding conditions, and the environment.

Of the 26 possible looping topologies and 8 possible tetrad arrangements available based on the glycosyl bond angles orientations, there are over possible unimolecular conformations for the simplest quadruplex The formation of multistranded structures further increases the possible conformational space.

Furthermore, the conformational polymorphism observed under solution conditions does not adequately reflect the conditions in vivo, including crowding, specific protein binding and the fact that such sequences are closed at one telomeres or both ends internal DNA sites such as promoter regions 29 , Polymorphism seems to be a common feature of G-rich sequences 1 , Whether this occurs in biological systems or only in vitro, it is important to understand the rules that determine the populations of the various structures under given conditions, especially for biotechnology applications.

Many quadruplex systems cannot be described structurally due to high complexity resulting from the simultaneous presence of multiple species. Ideally one would simply separate the various species present in solution in order to analyze them individually.

Unfortunately, for G-quadruplexes it is difficult to apply standard separation techniques because, as we show here, different folds of the same sequence often have nearly identical physical properties.

This may contribute to the inconsistencies in the reported structural and biophysical data for differently modified systems derived from a single sequence. There are several theoretically possible monomeric, dimeric or tetrameric G-quadruplex topologies for this sequence as shown in Figure 1.

In practice, it folds into at least eight different monomeric quadruplex structures under the same experimental conditions. Furthermore, a different species is produced by rapid cooling, indicating significant differences between kinetic and thermodynamic stability.

We will show that many of these species can be resolved by chromatography. We have characterized individual conformers by several biophysical techniques, and compared them both with one another and with the unresolved mixture.

We have demonstrated that each conformation has very similar hydrodynamic and electrophoretic properties, and similar thermodynamic stability, yet can be resolved by chromatography into species that have very distinct nuclear magnetic resonance NMR.

With one exception, the circular dichroism CD spectra could not discriminate among the different species. In the absence of sufficient resolution or incomplete theory for most commonly used biophysical techniques 36 , and because the G-rich oligonucleotides commonly adopt multiple conformations, analysis of the quadruplex system should begin from rigorous characterization by high information content, high resolution techniques such as NMR, before detailed analyses of other biophysical properties are initiated, as we have previously suggested The oligomer was shown to be a single chemical species by electrospray mass spectrometry, high performance liquid chromatography HPLC and electrophoresis.

The guanine in the linker region G14 of the sequence was not included in the series. The DNA samples were then refrigerated until use. For calibration purposes the column was run at a flow rate of 0.

Elution was monitored at A For DNA separation, the flow rate was altered to 0. Gels were stained with ethidium bromide Sigma-Aldrich, St. Samples of individual fractions were collected from the FPLC, combined, and concentrated using microcon YM-3 Millipore centrifugal concentrators.

CD spectra of samples having an A of 0. The spectral band-width was set to 2 nm and data pitch to 0. Spectra were recorded with a response time of 2 s. Three spectra were averaged, and the buffer baseline was subtracted.

The T m was estimated from the first derivative of the melting profile at nm. The baselines outside of the melting transition were assumed to be linearly dependent on temperature according to:.

These equations refer to single melting and unfolding transitions, which are the same if the system approaches equilibrium at each temperature. Non-linear regression analyses were carried out using Kaleidagraph Synergy Software.

Sedimentation velocity profiles were recorded of unfractionated and fractionated AS using a Beckman Optima XLA ultracentrifuge equipped with absorbance optical system and An Ti rotor.

All samples were dissolved in the FPLC buffer to working concentrations resulting in 0. Sedimentation coefficients showed little dependence on concentration under these conditions.

Equilibrium centrifugation provided an independent estimate of M. A value of 0. Under these conditions, all of the quadruplex converts to duplex 30 , and with a rate constant that largely represents the unfolding rate constant of the quadruplexes under the same conditions Both samples also had the same relative electrophoretic mobility, and appeared as a single band, as shown in Supplementary Figure S1.

Furthermore, the melting profile of the annealed oligonucleotide was very similar to that of the cooling profile, suggesting a reversible process at this heating and cooling rate.

The sedimentation velocity profile, c S Supplementary Figure S2 of the mixture shows two species with sedimentation coefficients of 2. The larger mass corresponds to a dimer. We conclude therefore that the oligonucleotide forms a mixture of stable monomeric and dimeric G-quadruplexes.

The frictional ratio of the monomers Equation 3 is close to unity, and given that nucleic acids are typically well hydrated 40 , 44 , 45 , this suggests that the monomeric species is highly compact, and behaves hydrodynamically as a nearly spherical object.

Although the biophysical data are consistent with the presence of a monomeric quadruplex species and a certain amount of dimer, these low-resolution techniques do not unequivocally discriminate the several possible folding topologies Figure 1 , and thus experimentally generate averages.

To reveal the real sample content requires high-resolution structural techniques, such as NMR. The frequency range of the imino proton resonances Figure 3 shows a chromatogram of the slowly annealed AS The first three fractions account for two-third of the total.

For comparison, a 10 kDa protein elutes at the same volume as fraction 2. However, the CD and sedimentation velocity profiles show that not only are these species quadruplexes, but also that they are monomers having a Stokes radius and mass the same as that of the other fractions, and thus are not breakdown products Table 1.

Remarkably, all of the fractions 1—5 show a very similar mobility to that of the parent mixture. As the fractions all have the same frictional ratio Table 1 this implies that the effective charge distributions are also similar.

Fraction 1 is a mixture of monomer and dimer species, and appears as a poorly resolved doublet by gel electrophoresis, with a slower band migrating at ca. Thus, electrophoresis does not necessarily resolve multiple isomeric species; a single band is not conclusive evidence of a pure component.

These spectra are nearly isodichroic at nm. These bands correspond to Cotton effects in the absorption spectra, which have a maximum at nm, and a shoulder at nm not shown. These are typical of G-quadruplex CD spectra 36 , As we will show, this species has a substantially different NMR spectrum from that of the other fractions.

The annealing curves for fractions 1—5 are extremely similar as expected Figure 4. Fraction 1 shows a broad overlap of resonances in the NMR spectrum Figure 5 , many in common with those of the mixture.

This species is known to be a mixture of a monomeric and dimeric quadruplex forms Table 1 and presented AUC data. Fractions 3 to 5 are all monomeric, and the imino proton resonances are sharp and relatively well resolved.

Nevertheless, both fractions 3 and 5 have roughly 30 GN1H resonances, indicating that these species too are mixtures. The best resolved spectra are those of fractions 2 and 4, which show a number of GN1H resonances similar to that expected for a monomeric species containing 17 GN1H.

Some of the GN1H in Fraction 4, however, are broad and ill-defined, and there are no GN2H resonances 9—10 ppm visible, suggesting a less stable species. This behavior is analogous to the human telomeric sequences 27 , The chromatographic fractions clearly correspond to different G-quadruplex species all derived from a single oligonucleotide chain.

Under slow annealing conditions the final populations should approach the equilibrium distribution, as indicated in Table 1. The free energy differences between these species are very small, i.

Moreover, they are kinetically very stable, as they do not interconvert on the timescale of the chromatography experiment or for extended periods in the NMR days. This suggests that the folding process from an unfolded strand can be represented by the following model.

In contrast, fraction 2 shows a single GN1H resonance not shown. As the relaxation properties and magnetization transfer function to be essentially the same for each conformation, we have volume integrated the peaks in each cluster, which were then used to estimate the relative populations of the species present.

Figure 6 shows at least eight distinguishable states for G8, which is comparable to the number resolved by chromatography. We have obtained similar results for other G resonances, implying that the conformational heterogeneity extends throughout the structure e.

The eight most abundant species have mole fractions ranging from 0. This corresponds to an equilibrium folding experiment, and the CD spectra maintain isodichroic points throughout the folding profile, despite the end state being a mixture of different species albeit most having similarly shaped spectra.

The apparent free energy of folding was We note that this free energy of folding is similar to that observed for globular proteins of the same molecular weight This is in agreement with the observation that the major species reach similar populations under slow annealing conditions.

The melting and annealing profiles of the unfractionated mixture are essentially superimposable cf. Figure 2 B and Table 1. In contrast, the individual fractions showed quite different annealing and melting profiles, as expected because the isolated fractions are not at equilibrium.

Furthermore, during melting the isolated fractions do not maintain an isodichroic point Supplementary Figure S5. Thus the starting point for melting is different for each fraction, though the end point is the same.

As the temperature is raised, a given fraction begins to unfold, and that unfolded portion will re-equilibrate with all of the other folded forms. For example, starting with pure fraction 2, as the temperature is increased so that a significant amount unfolds, some will re-equilibrate into all of the other fractions, which then also eventually melt; the concentration of these states is then expected first to increase and then decrease with increasing temperature.

The effect on the CD intensity will also be determined by the specific CD intensity of each contributing species. Over time, the quadruplex imino proton resonances decrease in intensity, with a concomitant increase in the intensity of the duplex resonances.

The complementary C-rich strand under these conditions showed no imino proton resonances, and the non-exchangeable protons were sharp, consistent with a flexible unstructured oligonucleotide data not shown.

The proton spectrum of the duplex form has GC: AT imino proton intensities in the ratio 2: Figure 8 B shows the time dependence of the area of the duplex resonances 12—14 ppm and of the quadruplex GN1H resonances The data cannot be fitted to a single exponential, and there are at least two classes of processes.

The biexponential fits returned rate constants of 0. An additional experiment carried out using a lower concentration of the complementary strand gave similar kinetics, suggesting that these rate constants do reflect the unfolding of the quadruplexes.

The most recalcitrant species still present at 66 h appears to decay considerably more slowly than this. Hence, the half-life of this quadruplex must be of the order 66 h under these conditions.

This implies that the kinetic stability of the different species varies over at least a fold range. As for other such intramolecular quadruplexes 42 , 51 , 53 , the AS quadruplexes are extremely kinetically stable, but not especially thermodynamically stable.

The annealing and melting profiles are deceptively simple, in part because at the lower temperatures, the folding and unfolding is not always close to equilibrium. Under these conditions, the distribution of folded forms approaches the expected thermodynamic distribution.

As the temperature is lowered further these become locked-in kinetically. In contrast, individual conformers isolated chromatographically are no longer at equilibrium, but exchange among conformers is very slow.

For a polynucleotide initially in the absence of cations, the first step may consist of rapid neutralization of backbone charge by added cation, resulting in collapse of extended polynucleotide to give an ensemble of compact hairpin structures.

Since the rate of complex formation depends on the fourth power of the monomer concentration, the actual time required for tetramerization is strongly dependent on the initial concentration of monomers and the nature of the cation and can vary from a few minutes to days.

On the other hand, formation of bimolecular quadruplexes is a second-order process and is relatively slower than unimolecular quadruplex formation. Different spectroscopic methods that probe different structural features may give different folding kinetics.

Some of these differences may result from the specific sequences or conditions [e. However, with the exception of the SPR data summarized above, the kinetic studies suggest that cation-driven quadruplex folding is a multistep process with detectable amounts of obligatory intermediates.

The intermediates are rate-limiting because relatively high-activation energies are required to produce conformational changes necessary for formation of complex topologies that may require one or more strand reversals and positioning of loops.

However, in conclusion, it is worth repeating the classical caveat of kinetics: The general principles of macromolecule stability are well known, , and for NA duplexes and to a lesser extent triplexes have been described in some detail, , , , — Although the same principles must apply to quadruplexes, the issue of the balance of forces for quadruplexes has not received much attention.

Short quadruplexes at least are significantly different from duplex and triplexes because of the nature of the H-bonding interactions, the stacking of G-quartets, and the involvement of specific ionic interactions direct inner spheres coordination as well as any nonspecific electrostatic effects of ionic interactions.

The description of the stability of any structure is best couched in terms of the free-energy contributions, as described earlier for duplex and triplex NAs.

Here each component is defined as follows: The bonds term arises from bond length and angle enthalpic factors, and el refers to electronic interactions including polarization and the exchange terms involved in, for example, the hypochromic effect in the bases.

In terms of computation, the standard parsing of the free energy of stabilization of a structure is couched in simple terms that can be loosely separated into the potential energy and the entropic contributions.

For a highly cooperative two-state folding transition and see below for recent studies concerning the cooperativity and number of intermediates, the potential energy terms can be described as a molecular mechanics potential such as Equation Generally, it is assumed that all individual components are independent and harmonic 51— Until recently, the polarizability of various groups was ignored amber.

The latter depends on the details of the model being used full atom discrete model with explicit solvent versus continuum model etc. The issue of the partial charges is also complicated and they have to be carefully calculated from a relatively high-level theory, and thus for isolated nucleotides.

However, the nucleotides are aromatic systems, and as such are quite polarizeable. If one assumes local i. A fully anisotropic polarizability a tensor quantity would be considerably more involved to implement.

Current versions of mechanics programs treat polarizability in a fairly simple manner — The treatment of hydrogen bonds can be explicit or implicit, depending on the implementation.

As the H-bond in biological system is largely electrostatic and van der Waal’s, these terms properly calibrated can be sufficient to account for the H-bond energy. However, H bonds in principle have a covalent character to them, and this can be detected by for example NMR methods 84—86 , Density functional theory DFT calculations have shown how the scalar contribution depends on distance and orientation of the donor and acceptor atoms These force fields do not account for optical effects that arise from the pi stacking, including the hypochromicity and CD.

The same mechanics force field can be used both for calculated a potential energy or in principle the difference between two states, see below. As the important determinant of stability is actually the free-energy difference between the state of interest and all other possible realizable states, the entropic components also need to be understood.

The configurational entropies of the folded and unfolded states for example can be calculated from long dynamics calculations or Monte-Carlo sampling. However, the largest differences in entropy are likely to arise from the solvation and electrostatic terms, as the numbers of associated water and counterions may be very different between the compact and extended states.

As the nature of a nonfolded G-rich DNA strand is unknown, and experimentally very difficult to evaluate ensemble of conformations, variable degrees of base—base stacking that depend on temperature and salt, such calculations are presently daunting.

For this reason, modeling stability generally looks at the neighborhood of the local energy minimum of the folded state to evaluate its resistance to substantial perturbation.

This is not thermodynamic stability, but may be relevant to kinetic stability. However, calculations of free energies associated with the folding landscape have been attempted 51 , Each quadruplex is associated with the formation of two H bonds per G, or eight per quartet.

The acceptors and donors in the strand state are probably hydrogen bonding to water, so the net change in the number of H-bonds is zero, and thus the enthalpy change from intramolecular H-bond formation is relatively small However, there may be a favorable entropy change due to the release of water into the bulk solvent.

As pointed out above, there may be a small net energy gain in the folded state from the covalent character of the NH:: N H-bonds formed 85 , as well as from change in the dipole interaction energy in a region of lower dielectric permittivity compared with full exposure to water.

Generally, however, the contribution of H-bonding to the free energy of stabilization is thought to be small, , The potential energy from van der Waals interaction in a well-optimized structure is large and negative, because of the very large number the small favorable interaction energies between pairs of atoms.

As each atom is essentially close packed, then any expanded structure in vacuo will result in a large net unfavorable energy. However, in a solvent the unfolding of a NA or protein is compensated by an essentially equal number of similar van der Waals interactions with the solvent molecules, such that the net stabilization energy is rather small This is likely to be roughly linear per stack added, though the actual net favorable energy can be offset by unfavorable strain from loop lengths, which is itself structure dependent 62 , 75 , , The stacking interactions are similar to those found in other NA structures, and likely account for a substantial part of the net stabilization free energy.

The most destabilizing component in NAs at neutral pH is the very unfavorable electrostatic interactions between the oxygen atoms in the phosphodiester bonds, which bear a formal charge of —1.

This is exacerbated when a NA strand folds, and this unfavorable energy needs to be offset by all other favorable interactions, or by neutralization by counterions.

In quadruplexes, the specific site binding of approximately one monovalent ion per quartet is one part of a favorable stabilization. In quadruplexes, there are two kinds of ion binding. Such ions interact as outer sphere complexes i.

The standard way of estimating number of ions released or absorbed on folding is to use thermochemistry. The melting temperature will depend on the concentration of salt if the charge density differences between the states:.

For NA unfolding, the formal charge arises for the phosphodiesters, i. The formal charge leads to a high intrinsic electrostatic free energy, which is unfavorable.

If the charge density exceeds a critical value, which depends on geometry as well as the number of charges, then ions will condense and neutralize the charge. The critical parameter for nonspherical objects is the so-called Bjerrum length.

Thus for a B-DNA polymer, about 0. In other words, a mer duplex will release The condensation model has been verified by experiment and extensive Poisson—Boltzmann calculations.

The latter have shown that short oligonucleotides deviate somewhat owing to so-called end effects the ends of a duplex have a lower charge density than those in the interior suggesting that these theoretical values would overestimate the experimental values for short oligonucleotides, More recently, Manning, has shown that even a spherical charge distribution will condense ions, depending on the actual charge density, and also on the ionic strength, albeit less than a cylinder.

Conversely, if a folded, intramolecular fold such as a quadruplex were to condense less ions than the unfolded state, then the salt-dependent stability would be the reverse of the duplex-strand transition, i.

Unfortunately, there seem to be no Poisson—Boltzmann calculations carried out on small, well-defined quadruplexes, for which one might imagine that the specific of topology and thus charge density would be important.

The uptake per mole of formal negative charge varied from 0. However, not all of the phosphates are involved in the G-quartet stacks i. If the uptake of ions is normalized, rather arbitrarily, to the number of nonloop phosphates, the uptake is much larger, i.

These are still low numbers when one takes into account also that these structures are binding 2—3 ions specifically, i. Again, the binding of two to three specific ions in the G-quartets would itself account for 0.

Although the discrepancies among the various sequences and methodologies are substantial, the number of ions taken up per quadruplex formed is comparable to the expected number of specific ions bound, suggesting that the ionic strength effects may be quite small compared with the more familiar duplex folding.

Given the uncertainties regarding ion condensation, it is unclear whether these numbers are merely fortuitous. Nucleotides exposed to solvent unfolded states are hydrated.

However, approximately four water molecules per nucleotide is released on unfolding of DNA duplexes, It is possible to determine the amount of water released or taken up during a transition by using osmolytes to affect the water activity, provided they can be shown to be true osmolytes and do not interact directly with either state of the solute of interest, Such studies have been carried out for duplex and triplex DNA It is also possible to combine electrostatics and hydration simultaneously by varying the concentration of salt, which alters water activity.

In DNA duplex unfolding, the stabilizing effect of increasing ionic strength is opposed by the decrease in water activity as the duplex is more hydrated than the strands, e.

The first term on the right-hand side of Equation 13 is the nonideality effect of salt on the DNA, the second term represents the decrease in water interaction due to replacement by salt, the third is the nonideality term from salt—water interactions and the final term is the electrostatic salt—salt nonideality effect.

The nonideality and cross terms in Equation 14 become especially important at higher salt concentrations, such as approaching the 1 M standard state often used in NAs thermodynamics where the polyelectrolyte contribution is zero, This is opposite to DNA duplex folding, which is associated with an uptake of water, Small molecule osmolytes thus stabilize the quadruplexes.

Further, other cosolvents such as primary alcohols that not only decrease the water activity, but also the bulk dielectric constant, were shown to increase quadruplex stability, which is opposite to the well-known effect of alcohols on DNA duplex solubility and thermodynamic stability.

This behavior was interpreted in terms of favorable electrostatics for folding. In a formal sense, the ion binding is described by a simple thermodynamic square as shown in Scheme Here if m, n are greater than 1, it is assumed that the cooperativity of binding is very high cf.

The temperature dependence of folding, as in UV melting for example, would be This becomes a linear function of when K 4 is large compared with, and K 2 is small compared with. The slope approaches n, the number of ions bound in the folded state.

In an optical titration, the observed signal depends on the specific absorption coefficient. The assumption that metal binding does not in itself affect the absorption coefficients of the nucleotides is for illustrative purposes only and represents the simplest case.

Equation 16 is a Hill equation and will show positive binding cooperativity of L at a fixed temperature. In most cases, the values of K 1 would be large, so the apparent dissociation constant for e.

Under such conditions, the Hill coefficient n is an indicator of the number of ions bound during folding These affinities are apparent as they are accompanied by folding, as in Scheme 2. As the specific ion binding to the unfolded state is also likely to be small, the stabilization due to specific ion binding should be substantial i.

This corresponds to 7. As oxygen ligands are replaced by oxygen ligands, one can envision that the enthalpy change of pure ion binding is relatively small. This further implies that under appropriate circumstances, the population of the unfolded state in the absence of cation could be significant.

This in general accord with the kinetics of formation of the human telomere quadruplex initiated by sodium or potassium in which cation binding is highly cooperative, both under equilibrium conditions and in the early phase of the approach to equilibrium.

Nevertheless, this analysis is highly simplified, and really reflects our current lack of understanding of the energetics of quadruplex formation and stability. Hud and coworkers 31 have recently summarized the literature on ion binding to G-quadruplexes.

The difference in stability between sodium and potassium forms is well documented, though there is a tendency for these ions stabilized different structures, in which the coordination stereochemistry of the ions also differ cf.

Experimentally, the difference in free energy between potassium and sodium binding is of the order 1. This is in agreement with calculations that indicate essentially free mobility of small ions e.

An actual parsing of the component energies has yet to be achieved. In Quadruplex topologies and structures section, we alluded to the restrictions imposed by loop length on possible topologies and by implication the contribution to the overall stability of different quadruplex structures.

Although we have argued so far that bases stacking and specific ion binding may be the dominant stabilizing interactions, the length and sequence of these loops is important.

Similarly, replacing nucleotide with nonnucleotide linkers had substantial effects on both stability and folding kinetics Loop length clearly influences the energetics of quadruplexes, because changing the length from 1 nt to 2 or 3 is associated with a change on overall fold In general, one would expect the stability to increase with the number of stacks.

Similarly, in the series GnT2 4 , the free-energy change was also linear in n, apart from one anomaly, with a slope of 1. Interestingly, the apparent number of potassium ions taken up on folding barely increased with n from 2.

The presence of the T2 loops was suggested to allow for different topologies antiparallel rather than parallel Nonspecific ion dependencies might also account for some of the anomalous estimates of the number of ions bound, as may the annealing history of the nucleotides e.

In any event, this all points to the complexity of dealing with quadruplex structures, and the need for simple but rigorous methods to establish what structures are present and the mole fractions under the experimental conditions when multiple conformations are present.

Because thermodynamics refers to changes in state functions, all thermodynamic considerations must include nature of the unfolded state. Even very high quality energy calculations on a particular structure imply little about net thermodynamic stability.

The rather dense purine sequences of G-tracts suggest that the single stranded state at normal temperature and salt conditions is unlikely to be remotely like an extended strand or a random coil; significant base stacking of nearest neighbors is to be expected.

Telomeric DNA can be prevented from forming quadruplex structures by ensuring that there are no high Z cations present. A macrocation such as the tetramethylammonium TMA ion will also prevent quadruplex formation better than Li, because the ion is simply too large to fit into the cavity of the quartet.

However, its CD spectrum has a classical conservative exciton coupling shape, indicative of significant base stacking, but not consistent with G-quadruplex folded structures R. Thus, the strand state is by no means a random coil.

In principle, FRET could be used to assess the distribution of end-to-end distances in the unfolded state Such FRET experiments in TMA might provide useful information about the distribution of size and shape in the unfolded form, and thus the degree of stacking in the strand state.

Given the preceding brief overview, we now discuss the factors that determine formation of known structures, and what can actually form, in terms of kinetic versus thermodynamic control, and how these might be modulated by cellular environments.

NA folding is associated with altered hydration. The effect of water activity can be estimated using osmolytes to influence the water activity, and the data can be analyzed in terms of preferential hydration as described by Timasheff — and by Parsegian, The dehydration would stabilize quadruplexes compared with duplexes, which are more hydrated than the single strand and thus the quadruplex state.

This macromolecular matrix in itself disregarding for the moment any specific or nonspecific direct interactions can influence folding energy and kinetics as described by Minton, based on considerations of the coefficients of the virial expansion of state.

Both theoretically and experimentally, it is shown that the presence of noninteracting matter will favor the more compact state, i. Note that this would synergize the effects of water activity on hydration.

Sugimoto, have used crowding conditions in vitro to determine the influence on conformation and stability. They found that the crowding conditions caused a change in the structure of the quadruplex, owing to different excluded volumes cf.

Interestingly, as the crowding also decreases the water activity, it was argued that crowding conditions stabilized the parallel G-quadruplex structure because it releases water on formation Separating the contributions from crowding, solvation and electrostatics is complex, as they are not linearly independent parameters [cf.

A degree of separation may be achieved by using a range of small molecule osmolytes to probe water activity effects, and large, truly physically excluded noninteracting polymers for crowding effects in comparison with the small molecules, In vivo this is much more complex, because the proteins present in the nucleus are clearly not neutral particles, but rather a collection of architectural, structure and sequence selective molecules.

The folding of a single-strand G-rich overhang such as the one that exists in telomeres is a very different proposition from the formation of a G-quadruplex in an internal position, such as identified in numerous promoters 60 , This area of research has been recently reviewed In the former case, the folding is free to occur from an unconstrained end, and does so on a potentially stabilizing nucleus of double-stranded B-like DNA In contrast, from the point of view of energetics, the internal G-quadruplexes require the separation of the DNA strands to form a loop very unfavorable followed by the G-rich strand forming a quartet while being tethered at both ends, leaving the complementary strand as a loop, or as an i-motif as shown in Figure 9.

Based on estimates of the energies of formation of these structures, , , it is possible to calculate the cost of formation of such a structure, as follows. The T m depends on the fraction GC and the ionic strength as The values of T m for three different GC contents and three ionic strength values are given in Table 3.

The ionic strength covers the range used experimentally and the average intracellular monovalent ion content. Note that the duplexes are stabilized by salt less than the quadruplexes.

The van’t Hoff enthalpy estimates from ref. Extrapolation to a common reference temperature can also lead to error. To some extent, this energy cost might be offset if the complementary strand were to form a stable structure, such as the i-motif — However, this structure is at best marginally stable at physiological pH and temperature though the actual state of protonation of the cytosines in the nucleus is far from clear.

The reason for this is that the i-motif involves H-bonding between cytosines, on which one of the pair must be protonated on N3. Thus, these structures are stable only at low pH This does not include the unwinding stress and junction with neighboring duplex, although it is possible to build models, as yet unrefined, that simultaneously incorporate a G-quadruplex and an intramolecular i-motif on the complementary strand 65 , Figure 9.

Experimentally, the duplex wins over the quadruplex, , , as expected on thermodynamic grounds see above. An alternative is that a quadruplex becomes stabilized by a protein or other ligand, or that the single-stranded complement is similarly stabilized by a binding partner, such as a DNA.

RNA hybrid as in Escherichia coli 8 , which is typically a rather short hybrid duplex that is important in transcription initiation, or a ss binding protein. Another alternative is that supercoiling stress generated by transcription might supply this energy even transiently, though one might expect the less AT-rich regions to form a bubble in preference to a GC-rich sequence, unless again there are additional proteins that could stabilize on the other strand.

It is clear that such a structure will not spontaneously form without some help. It has been suggested that supercoiling such as during transcription can propagate the necessary stress back toward the promoter to cause unwinding of sequences, and thus permit the formation of the G-quadruplexes It is unclear why AT-rich sequences would not melt in preference to the GC-rich sequences, given the very large difference in stability.

Furthermore the applied torsional stress would surely change as the transcription proceeds, so that at some point the G-quadruplex would revert to the duplex state.

However, as the kinetics of reversion are typically very slow, this might not be possible unless other proteins were to accelerate the kinetics. Nevertheless, the implication was also that the system operates far from equilibrium, implying that an understanding of the kinetics is extremely important.

Indeed, it has been proposed that specific DNA-binding proteins bind to the single strand region in the torsionally stressed Myc promoter Risitano showed that in fact the ds moiety of the human telomeres is preferentially double stranded at physiological pH, but an increase the number of G-stacks favored the quadruplex over the duplex such as for the c-myc G-rich sequence, even in the presence of a large excess of the complementary C-rich strand The excess C-rich strand 5- to fold is thus in very large excess of the values of K 1 K 2 , and this should easily outcompete the quadruplex for all sequences studied, in agreement with, , For an internal loop, the penalty will be higher than for a free duplex Major arguments in favor of G-quadruplex formation in vivo include the ready ability of the G-rich sequences widely found in genomic DNA, as single strands spontaneously form a stable quadruplex structures in solution.

Furthermore, the prevalence of such G-potential sequences that far exceeds chance is argued to be preserved for a functional purpose. The latter argument has considerable weight but not specifically for the formation of any particular structure.

As discussed earlier, thermodynamic arguments favor the telomere as the most likely place to find G-quadruplexes in vivo. Such a structure would however protect the end of the chromosome from unwanted dimerization which would interfere with replication and with subsequent accurate chromosome segregation during mitosis, i.

It is experimentally very difficult to demonstrate the presence on any specific DNA structure in vivo and even harder to prove its absence! Maizels 12 and Bryan 14 have recently summarized the evidence for the existence of quadruplex telomeric G4 DNA in vivo [see Kipling, pp.

An overview of the differences in complexity of telomere replication among various organisms is given by Gilson and Geli The best evidence exists for ciliates such as Oxytricha and Stylonychia which have a very different kind of chromatin than mammals and a very large number of telomeres.

The fundamental paper by Schaffitzel et al. These two antibodies showed no significant affinity for any other kinds of DNA or RNA, or other polyanions although no proteins were tested. Only the latter showed a positive result in the indirect immunostaining technique on isolated nuclei from Stylonychia lemnae, and then only in the vegetative state i.

Indeed, the multiple chromosomes from the vegetative state tend to aggregate end-to-end in this organism, via the intermolecular G-quadruplex, and this may explain why the in vivo chemical modification experiments failed to detect quadruplex in these organisms, For subsequent replication, this complex must be resolved.

It is worth noting that the observed rate constant for the approach to equilibrium as opposed to the unidirectional rate constants and thus the half-life for formation and dissociation of a structure are the same.

Consider the simple intramolecular folding mechanism:. If one were to start the reaction with adding ion to unfolded nucleotide, the formation of Q will proceed exponentially toward equilibrium, according to:.

In contrast, the spontaneous unfolding from Q will occur with the same rate constant k obs provided that there is nothing to mop up the resulting strand or ions. Only the initial rates differ in the two directions.

Anything that binds Q will slow down the rate of approach to equilibrium, and similarly anything that sequester U will do so too, as the rates depend on the concentration of U and Q.

A major conclusion, ultimately based on known in vitro properties of G-rich oligonucleotides, is that if quadruplexes are formed, they need to be resolved by proteins, and cells appear to devote considerable effort to prevention of G-quadruplex formation, because they are too stable for replication or transcription.

Of the 21 proteins listed as being able to bind to quadruplex DNAs in vitro, only one quadruplex was a fold-back structure Many other proteins can be shown in vitro to have affinity for such structures, but their functional significance is unclear.

Recently, it has been argued that only the extreme end of vertebrate telomeres form quadruplexes, and a protein proposed to be needed for human telomere unfolding has been characterized Much of the evidence for quadruplex formation in mammalian DNA in vivo is generally circumstantial and to date no high selectivity antibodies for human quadruplex structures are available.

Quadruplex binding ligands typically have rather modest affinity and selectivity even for the small numbers of structures tested, and do not always bind in the manner expected or designed, , New developments in quadruplex-binding ligands show substantially higher affinity and selectivity for the human telomere sequence, and these might be useful for such experiments, with the caveat that the ratio of nonquadruplex DNA to potential quadruplex DNA is very large, and that selectivity also needs to be assessed against protein binding and a wide variety of alternative DNA structures.

The biology of telomeres was summarized in a recent review regarding their need for maintenance, and what happens when the length is not maintained This is called the t-loop mechanism — Although the length of the telomeres is much greater than the persistence length of DNA at physiological salt concentrations, opening of an internal loop within the duplex and forming in essence an intermolecular duplex is, as explained above, energetically unfavorable.

In fact, it was observed that in the absence of a stabilizing factor, the efficiency of t-loop formation was very low; only substantial formation of a loop structure was observed in the presence of the telomere specific protein TRF2 Indeed, the loop model does not formally require strand invasion, only that there is some interaction stabilized by for example protein.

Indeed, it has recently been argued that just the single strand-overhangs form a t-loop stabilized by what amounts to a dimeric G-quadruplex However, this is not in agreement with original models based on large loops that imply circularization of the lengthy double-stranded portion of the telomere.

Although the binding energy for a loop of this size would be small if the DNA were naked, the entropic cost could be quite substantial, as the probability of finding a small number of configurations in which the end is close to a specific part of the telomere is small compared with all the other possible configurations We note that the chromatin rearrangement implies connection of the physical problem to metabolism via ATP hydrolysis and acetylation events.

This also begs the question as to the remarkable length variation of telomeres, even in mammals. It is clear from the foregoing that despite the extensive efforts expended to date, we still have a very limited view of what determines the folding and relative stability of different quadruplex structures, and how fast they form.

Even worse, for the most part the relationship to in vivo formation is even murkier. The latter is a consequence of at least two factors. First is that in vitro experiments concentrate on simple, relatively well-defined systems under conditions that typically do not approximate those found in vivo, as described in part in Discussion section.

Furthermore, it turns out that even the short intramolecular complexes are surprisingly complex, and detailed analysis is hampered by the presence of multiple conformations and slow folding kinetics.

Nevertheless, the basic principles of the energetics of G-quadruplex formation do need to be considered in any biological model, as well as the question as to where the energy comes from.

Thus, the thermodynamics and kinetics of small oligonucleotides have value in that it is possible to examine them in great detail under a wide variety of well-controlled conditions.

This allows one to sample the possible range of behaviors within a realistic range energies and timescales, which ultimately must be relevant to the biological conditions. It appears that naked G-potential DNA alone has physical properties that are not commensurate with some of the proposed biological functions and thus requires protein binding to provide the energy to manipulate these properties.

It is further notable that some of the proposed functions of such DNA, such as found in promoters, may need to be coupled to unwinding events, and operate far from equilibrium. Under these conditions an understanding of kinetics is essential.

We have argued that the specific ion binding and general electrostatics are a critical component for the energetics, and folding rates of G-quadruplexes, that distinguish them from most other DNA or RNA folds.

However, there are disturbing variations in basic thermodynamic properties that need to be addressed to establish whether these variations arise from experimental artifacts or there are real differences that are sensitive to conditions and small variation in sequence.

To accomplish this goal, we believe that an agreed upon and reliable set of rapid and inexpensive techniques is needed to determine the topology of any quadruplex, and assess its purity.

This is likely to come from a combination high-resolution spectroscopic and hydrodynamic techniques, such as NMR and AUC. First, methods are needed for rapid assessment of the topological structure s that is are present and whether the state is unique and thus can be manipulated by variation of conditions until it is.

This is a prerequisite for subsequent structural and functional analysis, purely as an analytical quality control. Second, it would make it possible to prepare a standardized set of oligomers of known structures to determine whether even simpler methods that exist can make useful distinctions among possible structures database approach.

To this end, we propose that a consortium be established to address major unresolved questions, or at least enlighten the present authors who confess having more questions than answers.

Some of these are listed below. Finally, there is a substantial literature on targeting G-quadruplexes for possible anticancer drug development 18 , 60 , which is beyond the scope of this review.

However, the physical principles apply equally to this area. This work was funded in part by NIH grant No. Funding to pay the open access charge for this article has been waived by NAR. We thank Paula J.

Bates and a reviewer for a critical reading of the article and valuable comments. Your ideas and opinions can help provide a better service for the life sciences research community. Europe PMC requires Javascript to function effectively.

The snippet could not be located in the article text. This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article. Published online August Brad Chaires, 1 Robert D.

Gray, 1 and John O. This article has been cited by other articles in PMC. Abstract In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function.

Chemical structures of G-quartets and quadruplexes. A Anticonformation top left and syn conformation top right of guanosine. B Inosine left and 7-deazaG right variations.

C G-quartet with metal ion coordination to GO6. In particular, we attempt to address the following questions regarding quadruplex formation that are directly amenable to experimental and computational methods: What are the possible structures of G-quadruplexes?

Stacked quartets with coordinated monovalent ion. Overview of possible and actual structures Even within the context of a small sequence space, the possible structural diversity of folding topologies of quadruplex structures is high 32 , Why is it so difficult to force a distinct topology?

Topologies give rise to radically different structural appearance. Methods of determining structures There are many approaches, both direct and indirect, to determining conformations of macromolecules at various levels of resolution.

Atomic resolution There are three main methodologies in use to assess the 3D structures of quadruplexes at atomic resolution: Spectroscopy The electronic spectroscopies have long been used to characterize the structures of quadruplexes 60 , as well as provide a convenient sensitive signal for monitoring transitions or ligand binding.

Absorbance and CD spectra. Use of fluorophores The 2-aminopurine is a fluorescent base that is a common replacement for A or G and is relatively unperturbing except in a G-quartet in this instance.

Hydrodynamics Hydrodynamic techniques such as sedimentation velocity and translational diffusion measurements, as well as those techniques that supply information about the rotational diffusion e.

Electrophoresis Electrophoretic mobility is commonly used to assess the number of states present and the kinds of folded structures that may be present Chemical modification Inosine or 7-deaza-dG Figure 1 substitution for G is expected to disrupt hydrogen bonding and therefore the stability of quadruplexes, The basic thermodynamic relationships that are relevant to quadruplex stability can be summarized as: Thermodynamic methods Temperature variation By far, the commonest thermodynamic variable used for characterizing NAs is temperature.

Thermal denaturation monitored by spectroscopic methods Spectroscopic methods for monitoring melting rely on there being a distinct difference in spectroscopic properties between the folded and unfolded states, and that there is preferably a linear dependence of the signal on concentration, i.

Thermal unfolding curves for the human intramolecular quadruplex. Whole-spectra melting data and the test of the two-state assumption. Calorimetric melting differential scanning calorimetry Differential scanning calorimetry DSC 19 , 20 , in which differential heat capacity is measured as a function of temperature, offers a method for measuring the thermodynamics of G-quadruplex denaturation as directly as possible.

Some representative results Table 2 shows some representative results for denaturation studies of the human telomere quadruplex structure obtained by van’t Hoff analysis of spectroscopic data.

Multiple conformations Numerous structures can form in vitro 63 depending on the conditions, and may coexist. Within the context of a spectroscopic melting study, these two models differ as follows: Thermal profiles for two folding pathways.

The populations of states in two possible pathways as described in the text was calculated. Model i Two species connected by unfolded state: Kinetics of formation and dissociation of quadruplexes There are two major reasons for determining the kinetics of formation and dissociation of quadruplexes.

Kinetics methods The kinetics of G-quadruplex folding can be broadly divided into three types depending on the number of individual polynucleotide molecules contributing the quadruplex: Slow mixing Han et al.

Analysis of thermal hysteresis If the rate of heating or cooling is faster than the rates of interconversion between the folded and unfolded states, then the folding or unfolding lags behind, leading to noncoincident heating and cooling curves.

Surface plasmon resonance Unimolecular folding of telomeric sequences has also been studied by SPR. Temperature jump Relaxation methods developed by M. Stopped flow kinetics Stopped flow has a long history in enzyme kinetics, but has not been used for NA folding kinetics.

SVD of the wavelength dependence of the binding isotherms indicated that folding generally proceeds for ODN1 and ODN2 through at least one intermediate according to the Scheme 1: H-bonds Each quadruplex is associated with the formation of two H bonds per G, or eight per quartet.

Generalized van der Waals The potential energy from van der Waals interaction in a well-optimized structure is large and negative, because of the very large number the small favorable interaction energies between pairs of atoms.

Ion binding and solvation Nonspecific ion binding and hydration In quadruplexes, there are two kinds of ion binding. The melting temperature will depend on the concentration of salt if the charge density differences between the states: Specific ion binding In a formal sense, the ion binding is described by a simple thermodynamic square as shown in Scheme 2: The apparent dissociation constant for unfolding would be.

Free energy of ion exchange Hud and coworkers 31 have recently summarized the literature on ion binding to G-quadruplexes. Number of quartets and length of loops In Quadruplex topologies and structures section, we alluded to the restrictions imposed by loop length on possible topologies and by implication the contribution to the overall stability of different quadruplex structures.

Nature of the unfolded state Because thermodynamics refers to changes in state functions, all thermodynamic considerations must include nature of the unfolded state. Intragene quadruplex formation The folding of a single-strand G-rich overhang such as the one that exists in telomeres is a very different proposition from the formation of a G-quadruplex in an internal position, such as identified in numerous promoters 60 , Formal model for formation of an internal G-quadruplex or other intramolecular structure.

Duplex DNA with a G-quadruplex potential sequence top unwinds and histones redistribute, leading to base-pair dissociation and formation of an open loop middle. Do quadruplex structures actually exist in vivo?

Consider the simple intramolecular folding mechanism: T-loops The biology of telomeres was summarized in a recent review regarding their need for maintenance, and what happens when the length is not maintained Develop reliable methods of rapid determination of topology of new sequences.

This entails generating a set of authenticated standards that can be reproduced according to specific procedures. The use of nonnucleotide linkers in comparison with nucleotides to establish contributions from loops to CD.

Investigate in greater detail the nature of the unfolded ensemble of states, perhaps using methods outlined in the text. This is essential to understand thermodynamic stability.

Determine the role of electrostatics in stabilization and kinetics of G-quadruplex structures. This will require additional methods such as capillary electrophoresis and careful salt-dependent analysis of the thermodynamics to establish the difference between nonspecific and specific ion binding, as well as the interplay with hydration.

Detailed calculations of the ionic contributions are also required to interpret such data. The electrostatics relate closely to issues of electrophoretic mobility. A systematic computation of CD for varied twist and wedge angles and induced CD of loop bases and see 2.

Develop structure-specific antibodies that can be used both for quality control, and also for probing the possible presence of structures in higher eukaryotes under different cellular conditions.

Apply novel methodologies to assess kinetic stabilization such as single molecule force measurements as described for RNA, Develop a realistic but experimentally tractable DNA system for telomeres and promoter G-quadruplex forming sequences that include nucleosome assemblies, and are compatible with optical and hydrodynamic approaches for example.

This is an area where a dialogue between the physical biochemists and biologists would be especially valuable to ensure that the problem is defined appropriately in a biological context.

Conflict of interest statement. Huppert JL, Balasubramanian S. Prevalence of quadruplexes in the human genome. G-quadruplexes in promoters throughout the human genome.

Beginning to understand the end of the chromosome. Chan S, Blackburn EH. Switching and signaling at the telomere. Lingner J, Cech TR. Telomerase and chromosome end maintenance. Oxford University Press; A human nuclease specific for G4 DNA.

Dynamic roles for G4 DNA in the biology of eukaryotic cells. Quartets in G-major — the first international meeting on quadruplex DNA. Oganesian L, Bryan TM. Physiological relevance of telomeric G-quadruplex formation: Extension of G-quadruplex DNA by ciliate telomerase.

Structure of the biologically relevant G-quadruplex in the c-MYC promoter. NMR methods for studying quadruplex nucleic acids. Gilbert DE, Feigon J. Natural and pharmacological regulation of telomerase.

Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids. Kinetics of tetramolecular quadruplexes. Model quadruplex interactions with a cationic porphyrin.

Nucleic Acid-Metal Ion Interactions. Royal Society of Chemistry; A topological classification of G-quadruplex structures. Nucleosides Nucleotides Nucleic Acids. Webba da Silva M. Geometric formalism for DNA quadruplex folding.

Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter. Crystal structure of parallel quadruplexes from human telomeric DNA. Yang Y, Patel DJ.

Structure of the intramolecular human telomeric G-quadruplex in potassium solution: The crystal structure of a parallel-stranded guanine tetraplex at 0. Biophysical Tools for Biologists: Vol 1 in Vitro Techniques; pp.

Biophysical and biological properties of quadruplex oligodeoxyribonucleotides. Nuclear magnetic resonance studies of drug-DNA complexes in solution. Sponer J, Spackova N. Molecular dynamics simulations and their application to four-stranded DNA.

Molecular dynamics simulations of guanine quadruplex loops: Advances and force field limitations. Free-energy component analysis of 40 protein-DNA complexes: Formation pathways of a guanine-quadruplex DNA revealed by molecular dynamics and thermodynamic analysis of the substates.

Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine. Towards a molecular dynamics consensus view of B-DNA flexibility. The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents.

Not so crystal clear: Effect of G-tract length on the topology and stability of intramolecular DNA quadruplexes. Structural diversity and extreme stability of unimolecular Oxytricha nova telomeric G-quadruplex.

Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene. Conformation and circular-dichroism of DNA. Circular-dichroism of polynucleotides — dimers as a function of conformation.

Sreerama N, Woody RW. Numerical computer methods, Pt D. Measured and calculated CD spectra of G-quartets stacked with the same or opposite polarities. A hitchhiker’s guide to G-quadruplex ligands.

Exceptionally slow kinetics of the intramolecular quadruplex formed by the Oxytricha telomeric repeat. Risitano A, Fox KR. Stability of intramolecular DNA quadruplexes: Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes.

Dale RE, Eisinger J. Intramolecular distances determined by energy-transfer — dependence on orientational freedom of donor and acceptor. Principles of Fluorescence Spectroscopy.

Structure and function in rhodopsin: Structure and function in rhodopsin.

More recent tests utilizing multiple wavelength data have appeared. A dual – wavelength parametric test for a two-state denaturation transition monitored by spectroscopy was described In this test, data obtained at two different wavelengths are plotted against one another.

For a two-state transition, such a plot should be strictly linear. Deviations from strict linear behavior signal that the denaturation process is not two-state, and likely has intermediate states that are significantly populated.

Singular value decomposition SVD provides an additional test of the two-state assumption, With modern diode array spectrophotometers, it is easy to collect entire spectra as a function of temperature, instead of single wavelength data.

A set of spectra as a function of temperature defines a 3D surface that is easily converted to a matrix. SVD of the matrix rigorously enumerates the number significant spectral species required to account for the spectral changes.

For a two-state transition, there should be only two significant spectral species, corresponding to the folded and unfolded forms. Any number of species greater than two indicates a violation of the two-state assumption, and signals the presence of intermediates.

SVD or a similar multivariate analysis method has been used to characterize the denaturation of G-quadruplex or other four-stranded structures, For both UV and CD datasets, there are clear deviations from strict linearity, a sure indication that the denaturation reaction is not a simple two-state process, and that intermediate states are populated to a significant degree and must be included in any reaction mechanism.

SVD analysis cannot be illustrated in a simple way, but the details of such an analysis are illustrated in refs 59 , , Unfortunately, it is enormously difficult to fit reliably transition curves to obtain derivative values of the primary thermodynamic parameters There is little that can be done to overcome these pitfalls in the analysis of spectroscopic transitions curves, but these difficulties must be acknowledged.

Calorimetry offers another tool that may overcome at least some of the problems. Differential scanning calorimetry DSC 19 , 20 , in which differential heat capacity is measured as a function of temperature, offers a method for measuring the thermodynamics of G-quadruplex denaturation as directly as possible.

The advantage of calorimetry is that total denaturation enthalpy values can be measured without recourse to any curve fitting or assumed models. Model-free calorimetric enthalpy values can thus be obtained directly from the primary data.

In addition, calorimetric thermograms can also be fit to particular thermodynamic model. Comparison of the model-free calorimetric enthalpies with such calculated model dependent enthalpies provides additional insight into the denaturation process, and in particular provides quantitative information about the cooperativity of the melting or the presence of intermediate states.

DSC studies are also plagued by baseline uncertainties. Processing of DSC data involves two types of baseline corrections. The first is subtraction of independently measured buffer baselines to correct for instrumental variances over the temperature range study.

This correction is straightforward and poses no difficulties. The second baseline correction involves choices similar to those discussed above, although in this case it is heat effects that contribute to baseline slopes and nonlinearities.

Even though calorimetry represents the gold standard for denaturation studies, it is not entirely without it own uncertainties, and investigators should describe and justify fully the choices that were made in baseline corrections.

Table 2 shows some representative results for denaturation studies of the human telomere quadruplex structure obtained by van’t Hoff analysis of spectroscopic data.

Data were selected for similar cation concentrations. The results are not comforting. Even worse, the free-energy change at K varies from the marginally stable 0.

These differences are unacceptably large, and the origins of the differences are by no means clear. The sequences used in these studies differed slightly, but it is difficult to believe that nucleotide end effects could exert such an enormous influence.

These data point to the need for additional studies to reduce the uncertainty in thermodynamic parameters. SVD was used to analyze temperature dependent circular dichroic spectra and to show that quadruplex denaturation was not a simple two-state process.

At least three species, corresponding to the folded, unfolded and one intermediate, were required in the reaction mechanism. DSC thermograms clearly showed two transitions.

The total calorimetric enthalpy for the overall denaturation process was dependent on KCl concentration, and varied from This study clearly indicates, at the least, that quadruplex denaturation is more complicated than was assumed in the studies shown in Table 2, and that intermediate states are significantly populated along the denaturation pathway.

Isothermal titration calorimetry ITC is most commonly used for binding studies, but a recent novel application used the method to study the enthalpy of G-quadruplex folding In this application, unstructured oligonucleotides were mixed with excess monovalent cation solutions in the calorimeter to monitor the total enthalpy of folding which includes any contribution from specific ion binding, see below.

By repeating the experiment at several temperatures, heat capacity changes could be estimated. Such a large heat capacity difference, comparable to that observed in small proteins, , would give rise to a large step in the DSC profile, which is not observed, and also imply cold denaturation at modest temperatures.

For the latter case, the temperature of maximum stability would be Numerous structures can form in vitro 63 depending on the conditions, and may coexist. If multiple conformations are possible, then they will form.

The question is one of populations. Many of the NMR analyses of quadruplexes have shown the existence of significant populations of alternative species 18 , 28 , 29 , 50 , and they have been detected or assumed to be present by other spectroscopic or thermodynamic approaches 30 , 62 , , — As described above, multiple states can be detected at equilibrium even where there is substantial cooperativity, where the states become substantially populated, or a wide range of probes is used.

For example, the UV-melting at a fixed wavelength may appear multiphasic in which case a fitting procedure according to an unfolding model is straightforward see below. Within the context of a spectroscopic melting study, these two models differ as follows:.

Allowing interconversion between N 1 and N 2 has no effect because there are only two degrees of freedom in this scheme. The populations of the species are given by:. These models differ in that the initial native points have in principle distinguishable properties.

Model 1 starts with a mixture of states that independently evolve toward the common end state, whereas model 2 implies that the two states present at low T i. N and I, populations determined by K 2 at sufficiently low temperature for example evolves through the intermediate state I.

Figure 8 shows a simple simulated comparison of an optical unfolding experiment in which there is an unfolded ensemble at high temperature, and a folded ensemble at low temperature.

The two models differ in the folded ensemble. The first model posits two alternative conformations that do not interconvert directly on any realistic experimental timescale they interconvert exclusively through the unfolded state.

The second model posits a sequential pathway with a fully native fold proceeding to the unfolded ensemble via an obligatory intermediate. In this simulation, these have been made equal at low temperature.

At low T, the distributions of I and N are determined by the equilibrium constant between them. Depending on the assumptions, the denaturation curve may appear either monophasic or biphasic. The simulated curves were fitted to the equation for a single transition, i.

All curves in this instance are well represented by a single transition, but the recovered enthalpies did not match the input values. For biphasic curves, of course one would fit a two-state transition of some kind for which the parameters would be better defined, assuming the correct model were chosen sic.

Model 1 corresponds to the situation described by Olsen et al. In fact, these considerations suggest approaches to producing states enriched in one or other structure.

Not only is the rate of cooling important 62 , but also the final temperatures may determine the distribution of structures present. This is determined largely by the enthalpy difference between the native folds.

Treating the observations as a single transition a common assumption yields unreliable thermodynamic parameters, as well as giving a false view of the unfolding process.

In principle, multiple wavelength observations i. Programs using SVD for example are well suited to this kind of analysis 64 and see above. Given these considerations, it is germane to question the meaning of some of the reported thermodynamic analyses.

There are two major reasons for determining the kinetics of formation and dissociation of quadruplexes. The first is that it is the only unambiguous means to obtain information about the mechanism of a complex reaction pathway.

As described above, under equilibrium conditions, it is not possible to discriminate among different pathways that connect initial and final states, and only detect intermediates if they become sufficiently populated with respect to the analytical techniques available.

The second major reason for measuring kinetics is to discover the general timescale of events, how they are modulated by conditions and additional factors such as small molecule ligands or possible binding proteins, to discover whether they are in fact commensurate with relevant biological time scales.

The kinetics of G-quadruplex folding can be broadly divided into three types depending on the number of individual polynucleotide molecules contributing the quadruplex: The kinetics of these processes has been comprehensively reviewed recently Generally, formation of bimolecular and tetramolecular quadruplexes is sufficiently slow at micromolar oligonucleotide concentration and physiological temperature that the progress of the reaction can readily be followed by conventional techniques.

Their studies indicated that the rate-limiting step in tetrameric quadruplex formation is a slow association of a pair of dimers that exist in relatively rapid equilibrium with single-stranded monomers.

If the rate of heating or cooling is faster than the rates of interconversion between the folded and unfolded states, then the folding or unfolding lags behind, leading to noncoincident heating and cooling curves.

This has been observed even in intramolecular quadruplex melting, especially at low salt concentrations, and makes thermodynamic analysis very difficult 62 , 75 , The kinetics of association and dissociation of multimeric quadruplexes have been extensively studied by Mergny’s laboratory using this approach.

This group utilized detailed analysis of the thermal hysteresis in nonequilibrium melting profiles results are summarized in reference This approach is capable in principle of giving both the association and dissociation rate constants as well as the apparent activation energies of both steps.

As summarized in Mergny’s review article, tetramolecular quadruplex association reactions generally exhibit negative apparent activation energies. Negative apparent activation energies or nonlinear Arrhenius plots are commonly observed in protein and NA folding and can result from any of several effects including i a temperature-induced change in the rate-limiting step s ; ii a change the heat capacity of the activated complex; or iii a temperature-induced change the ground state of the reaction, The kinetics of intramolecular folding and unfolding of G-quadruplexes is of interest because these studies can suggest possible folding pathways that involve rearrangements in secondary and tertiary structure that are necessarily obscured by the slow kinetics of multimolecular association processes.

The kinetics and mechanism of intramolecular folding processes including formation of simple structures such as hairpins as well as folding of more complex topologies such as ribozymes, Holliday junctions and riboswitches Intramolecular oligonucleotide folding has been investigated by a number of spectroscopic techniques that track different aspects of the dynamics of the folding—unfolding transitions.

Among these are multi-wavelength UV spectroscopy, changes in fluorescence emission of appropriately labeled oligonucleotides, FRET see above studies, and solid phase methods such as surface plasmon resonance SPR.

The method of single-molecule FRET has recently been utilized by two groups to assess the kinetics of G-quadruplex folding and unfolding and see Quadruplex topologies and structures section. FRET can either be monitored in solution yielding the average properties of the ensemble under a set of defined conditions or with a confocal microscope and suitable optics.

With the latter system, quite low e. One counts the number of molecules within a particular small range of transfer efficiencies and constructs a histogram of the distribution of molecules with different transfer efficiencies.

This serves as a convenient indicator of the distribution of conformational states Analysis of the temperature dependence of interconversion of the two populations allowed estimation of the thermodynamic parameters for each species in the presence of the two cations.

The kinetics of unfolding was assessed by trapping the unfolded species with the complementary oligonucleotide. Based on the analysis of the distance dependence of the FRET efficiencies in conjunction with model building, the authors suggested that one conformation is the parallel structure and the other is the antiparallel structure.

This again points to the prevalence of multiple conformations originating from the same sequence, which are condition and history dependent and see above. A complementary nt oligonucleotide that contained a tetramethylrhodamine-modified dT donor fluorophore was annealed to the G-rich oligonucleotide.

The two limiting folded states were suggested to be the parallel and antiparallel conformations. The time dependency of changes in FRET efficiency in single molecule experiments revealed that a fraction of the molecules rapidly switched between states, while others remained in the same FRET state for longer periods.

The structures of the short-lived states are not known, but the authors suggested that they are more compact that the unfolded states may consist of structures with less than the full complement of bound cations.

These states were suggested to consist of obligatory folding intermediates. FRET detection was also used to determine the unfolding kinetics of the human telomere, by trapping with a labeled peptide nucleic acid PNA i.

The excess PNA was shown to be zero order, so the observed transient could be attributed to the first-order unfolding of the quadruplex, followed by very much faster hybridization to the PNA.

Although the kinetics were biphasic, the data were analyzed in terms of a weighed mean lifetimes approach, giving an apparent opening rate constant of 0. Unimolecular folding of telomeric sequences has also been studied by SPR.

In this technique, the unfolded oligonucleotide is typically immobilized on a solid surface the sensor chip. A solution of a complementary oligonucleotide in the presence of a fold-inducing cation is pumped across the chip at a known flow rate.

The complementary oligonucleotide traps any unfolded immobilized G-rich oligonucleotide as a duplex. The rate of folding can be deduced in theory from the time dependence of signal changes occurring during the hybridization stage.

Once the complex is formed, the chip is washed with buffer alone to give the rate of unfolding as the cation dissociates. The corresponding rate constants for unfolding were 1. In general, as observed at equilibrium and by more refined studies in which folding is seen to be multiexponential see below, this ratio of apparent rate constant may agree with the true equilibrium constant only fortuitously.

In fact, the kinetically estimated equilibrium constant did not agree with values independently determined by equilibrium methods, by one to two orders of magnitude. Additional complications with the SPR approach, result from nonhomogeneous reaction conditions e.

The problems and solutions to these technical difficulties have been recently reviewed Thus, it may not be too surprising that the results obtained do not agree with the results obtained by more direct methods.

Relaxation methods developed by M. Eigen and coworkers are based on the return to equilibrium of a system after a small perturbation by a rapid change in a parameter such as pressure or temperature.

If the perturbation is sufficiently small, the response is linear in concentration. Specifically, for a single-step unimolecular reaction the relaxation rate constant is simply the sum of the forward and reverse reaction rate constants.

The approach is applicable in the neighborhood of the T m and provides a direct measure of the folding and unfolding rate at a given temperature. This approach was used in conjunction with hysteresis analysis to measure association and dissociation kinetics of the Oxytricha intramolecular quadruplex It was shown that the unfolding rate constant was extremely small, and that the association was characterized by negative apparent activation energy.

This latter requires a complex multistep process, such as the well-known zippering mechanism common in DNA duplex folding Stopped flow has a long history in enzyme kinetics, but has not been used for NA folding kinetics.

The titrations showed that folding is cooperative with respect to [cation] with Hill coefficients of 1. Cation half saturation concentrations for folding were 0. SVD of the wavelength dependence of the binding isotherms indicated that folding generally proceeds for ODN1 and ODN2 through at least one intermediate according to the Scheme The kinetics of folding was investigated using multi-wavelength stopped flow spectrophotometry The folding rates also tended to decrease with increasing temperature.

The following describes a possible mechanism that incorporates elementary steps from previous studies of oligonucleotide folding with the known structures of the final state and that is consistent with the kinetic data.

For a polynucleotide initially in the absence of cations, the first step may consist of rapid neutralization of backbone charge by added cation, resulting in collapse of extended polynucleotide to give an ensemble of compact hairpin structures.

Since the rate of complex formation depends on the fourth power of the monomer concentration, the actual time required for tetramerization is strongly dependent on the initial concentration of monomers and the nature of the cation and can vary from a few minutes to days.

On the other hand, formation of bimolecular quadruplexes is a second-order process and is relatively slower than unimolecular quadruplex formation. Different spectroscopic methods that probe different structural features may give different folding kinetics.

Some of these differences may result from the specific sequences or conditions [e. However, with the exception of the SPR data summarized above, the kinetic studies suggest that cation-driven quadruplex folding is a multistep process with detectable amounts of obligatory intermediates.

The intermediates are rate-limiting because relatively high-activation energies are required to produce conformational changes necessary for formation of complex topologies that may require one or more strand reversals and positioning of loops.

However, in conclusion, it is worth repeating the classical caveat of kinetics: The general principles of macromolecule stability are well known, , and for NA duplexes and to a lesser extent triplexes have been described in some detail, , , , — Although the same principles must apply to quadruplexes, the issue of the balance of forces for quadruplexes has not received much attention.

Short quadruplexes at least are significantly different from duplex and triplexes because of the nature of the H-bonding interactions, the stacking of G-quartets, and the involvement of specific ionic interactions direct inner spheres coordination as well as any nonspecific electrostatic effects of ionic interactions.

The description of the stability of any structure is best couched in terms of the free-energy contributions, as described earlier for duplex and triplex NAs. Here each component is defined as follows: The bonds term arises from bond length and angle enthalpic factors, and el refers to electronic interactions including polarization and the exchange terms involved in, for example, the hypochromic effect in the bases.

In terms of computation, the standard parsing of the free energy of stabilization of a structure is couched in simple terms that can be loosely separated into the potential energy and the entropic contributions.

For a highly cooperative two-state folding transition and see below for recent studies concerning the cooperativity and number of intermediates, the potential energy terms can be described as a molecular mechanics potential such as Equation Generally, it is assumed that all individual components are independent and harmonic 51— Until recently, the polarizability of various groups was ignored amber.

The latter depends on the details of the model being used full atom discrete model with explicit solvent versus continuum model etc. The issue of the partial charges is also complicated and they have to be carefully calculated from a relatively high-level theory, and thus for isolated nucleotides.

However, the nucleotides are aromatic systems, and as such are quite polarizeable. If one assumes local i. A fully anisotropic polarizability a tensor quantity would be considerably more involved to implement.

Current versions of mechanics programs treat polarizability in a fairly simple manner — The treatment of hydrogen bonds can be explicit or implicit, depending on the implementation.

As the H-bond in biological system is largely electrostatic and van der Waal’s, these terms properly calibrated can be sufficient to account for the H-bond energy. However, H bonds in principle have a covalent character to them, and this can be detected by for example NMR methods 84—86 , Density functional theory DFT calculations have shown how the scalar contribution depends on distance and orientation of the donor and acceptor atoms These force fields do not account for optical effects that arise from the pi stacking, including the hypochromicity and CD.

The same mechanics force field can be used both for calculated a potential energy or in principle the difference between two states, see below. As the important determinant of stability is actually the free-energy difference between the state of interest and all other possible realizable states, the entropic components also need to be understood.

The configurational entropies of the folded and unfolded states for example can be calculated from long dynamics calculations or Monte-Carlo sampling. However, the largest differences in entropy are likely to arise from the solvation and electrostatic terms, as the numbers of associated water and counterions may be very different between the compact and extended states.

As the nature of a nonfolded G-rich DNA strand is unknown, and experimentally very difficult to evaluate ensemble of conformations, variable degrees of base—base stacking that depend on temperature and salt, such calculations are presently daunting.

For this reason, modeling stability generally looks at the neighborhood of the local energy minimum of the folded state to evaluate its resistance to substantial perturbation.

This is not thermodynamic stability, but may be relevant to kinetic stability. However, calculations of free energies associated with the folding landscape have been attempted 51 , Each quadruplex is associated with the formation of two H bonds per G, or eight per quartet.

The acceptors and donors in the strand state are probably hydrogen bonding to water, so the net change in the number of H-bonds is zero, and thus the enthalpy change from intramolecular H-bond formation is relatively small However, there may be a favorable entropy change due to the release of water into the bulk solvent.

As pointed out above, there may be a small net energy gain in the folded state from the covalent character of the NH:: N H-bonds formed 85 , as well as from change in the dipole interaction energy in a region of lower dielectric permittivity compared with full exposure to water.

Generally, however, the contribution of H-bonding to the free energy of stabilization is thought to be small, , The potential energy from van der Waals interaction in a well-optimized structure is large and negative, because of the very large number the small favorable interaction energies between pairs of atoms.

As each atom is essentially close packed, then any expanded structure in vacuo will result in a large net unfavorable energy. However, in a solvent the unfolding of a NA or protein is compensated by an essentially equal number of similar van der Waals interactions with the solvent molecules, such that the net stabilization energy is rather small This is likely to be roughly linear per stack added, though the actual net favorable energy can be offset by unfavorable strain from loop lengths, which is itself structure dependent 62 , 75 , , The stacking interactions are similar to those found in other NA structures, and likely account for a substantial part of the net stabilization free energy.

The most destabilizing component in NAs at neutral pH is the very unfavorable electrostatic interactions between the oxygen atoms in the phosphodiester bonds, which bear a formal charge of —1.

This is exacerbated when a NA strand folds, and this unfavorable energy needs to be offset by all other favorable interactions, or by neutralization by counterions. In quadruplexes, the specific site binding of approximately one monovalent ion per quartet is one part of a favorable stabilization.

In quadruplexes, there are two kinds of ion binding. Such ions interact as outer sphere complexes i. The standard way of estimating number of ions released or absorbed on folding is to use thermochemistry.

The melting temperature will depend on the concentration of salt if the charge density differences between the states:. For NA unfolding, the formal charge arises for the phosphodiesters, i.

The formal charge leads to a high intrinsic electrostatic free energy, which is unfavorable. If the charge density exceeds a critical value, which depends on geometry as well as the number of charges, then ions will condense and neutralize the charge.

The critical parameter for nonspherical objects is the so-called Bjerrum length. Thus for a B-DNA polymer, about 0. In other words, a mer duplex will release The condensation model has been verified by experiment and extensive Poisson—Boltzmann calculations.

The latter have shown that short oligonucleotides deviate somewhat owing to so-called end effects the ends of a duplex have a lower charge density than those in the interior suggesting that these theoretical values would overestimate the experimental values for short oligonucleotides, More recently, Manning, has shown that even a spherical charge distribution will condense ions, depending on the actual charge density, and also on the ionic strength, albeit less than a cylinder.

Conversely, if a folded, intramolecular fold such as a quadruplex were to condense less ions than the unfolded state, then the salt-dependent stability would be the reverse of the duplex-strand transition, i.

Unfortunately, there seem to be no Poisson—Boltzmann calculations carried out on small, well-defined quadruplexes, for which one might imagine that the specific of topology and thus charge density would be important.

The uptake per mole of formal negative charge varied from 0. However, not all of the phosphates are involved in the G-quartet stacks i. If the uptake of ions is normalized, rather arbitrarily, to the number of nonloop phosphates, the uptake is much larger, i.

These are still low numbers when one takes into account also that these structures are binding 2—3 ions specifically, i. Again, the binding of two to three specific ions in the G-quartets would itself account for 0.

Although the discrepancies among the various sequences and methodologies are substantial, the number of ions taken up per quadruplex formed is comparable to the expected number of specific ions bound, suggesting that the ionic strength effects may be quite small compared with the more familiar duplex folding.

Given the uncertainties regarding ion condensation, it is unclear whether these numbers are merely fortuitous. Nucleotides exposed to solvent unfolded states are hydrated. However, approximately four water molecules per nucleotide is released on unfolding of DNA duplexes, It is possible to determine the amount of water released or taken up during a transition by using osmolytes to affect the water activity, provided they can be shown to be true osmolytes and do not interact directly with either state of the solute of interest, Such studies have been carried out for duplex and triplex DNA It is also possible to combine electrostatics and hydration simultaneously by varying the concentration of salt, which alters water activity.

In DNA duplex unfolding, the stabilizing effect of increasing ionic strength is opposed by the decrease in water activity as the duplex is more hydrated than the strands, e.

The first term on the right-hand side of Equation 13 is the nonideality effect of salt on the DNA, the second term represents the decrease in water interaction due to replacement by salt, the third is the nonideality term from salt—water interactions and the final term is the electrostatic salt—salt nonideality effect.

The nonideality and cross terms in Equation 14 become especially important at higher salt concentrations, such as approaching the 1 M standard state often used in NAs thermodynamics where the polyelectrolyte contribution is zero, This is opposite to DNA duplex folding, which is associated with an uptake of water, Small molecule osmolytes thus stabilize the quadruplexes.

Further, other cosolvents such as primary alcohols that not only decrease the water activity, but also the bulk dielectric constant, were shown to increase quadruplex stability, which is opposite to the well-known effect of alcohols on DNA duplex solubility and thermodynamic stability.

This behavior was interpreted in terms of favorable electrostatics for folding. In a formal sense, the ion binding is described by a simple thermodynamic square as shown in Scheme Here if m, n are greater than 1, it is assumed that the cooperativity of binding is very high cf.

The temperature dependence of folding, as in UV melting for example, would be This becomes a linear function of when K 4 is large compared with, and K 2 is small compared with. The slope approaches n, the number of ions bound in the folded state.

In an optical titration, the observed signal depends on the specific absorption coefficient. The assumption that metal binding does not in itself affect the absorption coefficients of the nucleotides is for illustrative purposes only and represents the simplest case.

Equation 16 is a Hill equation and will show positive binding cooperativity of L at a fixed temperature. In most cases, the values of K 1 would be large, so the apparent dissociation constant for e.

Under such conditions, the Hill coefficient n is an indicator of the number of ions bound during folding These affinities are apparent as they are accompanied by folding, as in Scheme 2.

As the specific ion binding to the unfolded state is also likely to be small, the stabilization due to specific ion binding should be substantial i. This corresponds to 7. As oxygen ligands are replaced by oxygen ligands, one can envision that the enthalpy change of pure ion binding is relatively small.

This further implies that under appropriate circumstances, the population of the unfolded state in the absence of cation could be significant. This in general accord with the kinetics of formation of the human telomere quadruplex initiated by sodium or potassium in which cation binding is highly cooperative, both under equilibrium conditions and in the early phase of the approach to equilibrium.

Nevertheless, this analysis is highly simplified, and really reflects our current lack of understanding of the energetics of quadruplex formation and stability.

Hud and coworkers 31 have recently summarized the literature on ion binding to G-quadruplexes. The difference in stability between sodium and potassium forms is well documented, though there is a tendency for these ions stabilized different structures, in which the coordination stereochemistry of the ions also differ cf.

Experimentally, the difference in free energy between potassium and sodium binding is of the order 1. This is in agreement with calculations that indicate essentially free mobility of small ions e.

An actual parsing of the component energies has yet to be achieved. In Quadruplex topologies and structures section, we alluded to the restrictions imposed by loop length on possible topologies and by implication the contribution to the overall stability of different quadruplex structures.

Although we have argued so far that bases stacking and specific ion binding may be the dominant stabilizing interactions, the length and sequence of these loops is important.

Similarly, replacing nucleotide with nonnucleotide linkers had substantial effects on both stability and folding kinetics Loop length clearly influences the energetics of quadruplexes, because changing the length from 1 nt to 2 or 3 is associated with a change on overall fold In general, one would expect the stability to increase with the number of stacks.

Similarly, in the series GnT2 4 , the free-energy change was also linear in n, apart from one anomaly, with a slope of 1. Interestingly, the apparent number of potassium ions taken up on folding barely increased with n from 2.

The presence of the T2 loops was suggested to allow for different topologies antiparallel rather than parallel Nonspecific ion dependencies might also account for some of the anomalous estimates of the number of ions bound, as may the annealing history of the nucleotides e.

In any event, this all points to the complexity of dealing with quadruplex structures, and the need for simple but rigorous methods to establish what structures are present and the mole fractions under the experimental conditions when multiple conformations are present.

Because thermodynamics refers to changes in state functions, all thermodynamic considerations must include nature of the unfolded state. Even very high quality energy calculations on a particular structure imply little about net thermodynamic stability.

The rather dense purine sequences of G-tracts suggest that the single stranded state at normal temperature and salt conditions is unlikely to be remotely like an extended strand or a random coil; significant base stacking of nearest neighbors is to be expected.

Telomeric DNA can be prevented from forming quadruplex structures by ensuring that there are no high Z cations present. A macrocation such as the tetramethylammonium TMA ion will also prevent quadruplex formation better than Li, because the ion is simply too large to fit into the cavity of the quartet.

However, its CD spectrum has a classical conservative exciton coupling shape, indicative of significant base stacking, but not consistent with G-quadruplex folded structures R.

Thus, the strand state is by no means a random coil. In principle, FRET could be used to assess the distribution of end-to-end distances in the unfolded state Such FRET experiments in TMA might provide useful information about the distribution of size and shape in the unfolded form, and thus the degree of stacking in the strand state.

Given the preceding brief overview, we now discuss the factors that determine formation of known structures, and what can actually form, in terms of kinetic versus thermodynamic control, and how these might be modulated by cellular environments.

NA folding is associated with altered hydration. The effect of water activity can be estimated using osmolytes to influence the water activity, and the data can be analyzed in terms of preferential hydration as described by Timasheff — and by Parsegian, The dehydration would stabilize quadruplexes compared with duplexes, which are more hydrated than the single strand and thus the quadruplex state.

This macromolecular matrix in itself disregarding for the moment any specific or nonspecific direct interactions can influence folding energy and kinetics as described by Minton, based on considerations of the coefficients of the virial expansion of state.

Both theoretically and experimentally, it is shown that the presence of noninteracting matter will favor the more compact state, i. Note that this would synergize the effects of water activity on hydration.

Sugimoto, have used crowding conditions in vitro to determine the influence on conformation and stability. They found that the crowding conditions caused a change in the structure of the quadruplex, owing to different excluded volumes cf.

Interestingly, as the crowding also decreases the water activity, it was argued that crowding conditions stabilized the parallel G-quadruplex structure because it releases water on formation Separating the contributions from crowding, solvation and electrostatics is complex, as they are not linearly independent parameters [cf.

A degree of separation may be achieved by using a range of small molecule osmolytes to probe water activity effects, and large, truly physically excluded noninteracting polymers for crowding effects in comparison with the small molecules, In vivo this is much more complex, because the proteins present in the nucleus are clearly not neutral particles, but rather a collection of architectural, structure and sequence selective molecules.

The folding of a single-strand G-rich overhang such as the one that exists in telomeres is a very different proposition from the formation of a G-quadruplex in an internal position, such as identified in numerous promoters 60 , This area of research has been recently reviewed In the former case, the folding is free to occur from an unconstrained end, and does so on a potentially stabilizing nucleus of double-stranded B-like DNA In contrast, from the point of view of energetics, the internal G-quadruplexes require the separation of the DNA strands to form a loop very unfavorable followed by the G-rich strand forming a quartet while being tethered at both ends, leaving the complementary strand as a loop, or as an i-motif as shown in Figure 9.

Based on estimates of the energies of formation of these structures, , , it is possible to calculate the cost of formation of such a structure, as follows. The T m depends on the fraction GC and the ionic strength as The values of T m for three different GC contents and three ionic strength values are given in Table 3.

The ionic strength covers the range used experimentally and the average intracellular monovalent ion content. Note that the duplexes are stabilized by salt less than the quadruplexes.

The van’t Hoff enthalpy estimates from ref. Extrapolation to a common reference temperature can also lead to error. To some extent, this energy cost might be offset if the complementary strand were to form a stable structure, such as the i-motif — However, this structure is at best marginally stable at physiological pH and temperature though the actual state of protonation of the cytosines in the nucleus is far from clear.

The reason for this is that the i-motif involves H-bonding between cytosines, on which one of the pair must be protonated on N3. Thus, these structures are stable only at low pH This does not include the unwinding stress and junction with neighboring duplex, although it is possible to build models, as yet unrefined, that simultaneously incorporate a G-quadruplex and an intramolecular i-motif on the complementary strand 65 , Figure 9.

Experimentally, the duplex wins over the quadruplex, , , as expected on thermodynamic grounds see above. An alternative is that a quadruplex becomes stabilized by a protein or other ligand, or that the single-stranded complement is similarly stabilized by a binding partner, such as a DNA.

RNA hybrid as in Escherichia coli 8 , which is typically a rather short hybrid duplex that is important in transcription initiation, or a ss binding protein. Another alternative is that supercoiling stress generated by transcription might supply this energy even transiently, though one might expect the less AT-rich regions to form a bubble in preference to a GC-rich sequence, unless again there are additional proteins that could stabilize on the other strand.

It is clear that such a structure will not spontaneously form without some help. It has been suggested that supercoiling such as during transcription can propagate the necessary stress back toward the promoter to cause unwinding of sequences, and thus permit the formation of the G-quadruplexes It is unclear why AT-rich sequences would not melt in preference to the GC-rich sequences, given the very large difference in stability.

Furthermore the applied torsional stress would surely change as the transcription proceeds, so that at some point the G-quadruplex would revert to the duplex state. However, as the kinetics of reversion are typically very slow, this might not be possible unless other proteins were to accelerate the kinetics.

Nevertheless, the implication was also that the system operates far from equilibrium, implying that an understanding of the kinetics is extremely important. Indeed, it has been proposed that specific DNA-binding proteins bind to the single strand region in the torsionally stressed Myc promoter Risitano showed that in fact the ds moiety of the human telomeres is preferentially double stranded at physiological pH, but an increase the number of G-stacks favored the quadruplex over the duplex such as for the c-myc G-rich sequence, even in the presence of a large excess of the complementary C-rich strand The excess C-rich strand 5- to fold is thus in very large excess of the values of K 1 K 2 , and this should easily outcompete the quadruplex for all sequences studied, in agreement with, , For an internal loop, the penalty will be higher than for a free duplex Major arguments in favor of G-quadruplex formation in vivo include the ready ability of the G-rich sequences widely found in genomic DNA, as single strands spontaneously form a stable quadruplex structures in solution.

Furthermore, the prevalence of such G-potential sequences that far exceeds chance is argued to be preserved for a functional purpose. The latter argument has considerable weight but not specifically for the formation of any particular structure.

As discussed earlier, thermodynamic arguments favor the telomere as the most likely place to find G-quadruplexes in vivo. Such a structure would however protect the end of the chromosome from unwanted dimerization which would interfere with replication and with subsequent accurate chromosome segregation during mitosis, i.

It is experimentally very difficult to demonstrate the presence on any specific DNA structure in vivo and even harder to prove its absence! Maizels 12 and Bryan 14 have recently summarized the evidence for the existence of quadruplex telomeric G4 DNA in vivo [see Kipling, pp.

An overview of the differences in complexity of telomere replication among various organisms is given by Gilson and Geli The best evidence exists for ciliates such as Oxytricha and Stylonychia which have a very different kind of chromatin than mammals and a very large number of telomeres.

The fundamental paper by Schaffitzel et al. These two antibodies showed no significant affinity for any other kinds of DNA or RNA, or other polyanions although no proteins were tested. Only the latter showed a positive result in the indirect immunostaining technique on isolated nuclei from Stylonychia lemnae, and then only in the vegetative state i.

Indeed, the multiple chromosomes from the vegetative state tend to aggregate end-to-end in this organism, via the intermolecular G-quadruplex, and this may explain why the in vivo chemical modification experiments failed to detect quadruplex in these organisms, For subsequent replication, this complex must be resolved.

It is worth noting that the observed rate constant for the approach to equilibrium as opposed to the unidirectional rate constants and thus the half-life for formation and dissociation of a structure are the same.

Consider the simple intramolecular folding mechanism:. If one were to start the reaction with adding ion to unfolded nucleotide, the formation of Q will proceed exponentially toward equilibrium, according to:.

In contrast, the spontaneous unfolding from Q will occur with the same rate constant k obs provided that there is nothing to mop up the resulting strand or ions. Only the initial rates differ in the two directions.

Anything that binds Q will slow down the rate of approach to equilibrium, and similarly anything that sequester U will do so too, as the rates depend on the concentration of U and Q.

A major conclusion, ultimately based on known in vitro properties of G-rich oligonucleotides, is that if quadruplexes are formed, they need to be resolved by proteins, and cells appear to devote considerable effort to prevention of G-quadruplex formation, because they are too stable for replication or transcription.

Of the 21 proteins listed as being able to bind to quadruplex DNAs in vitro, only one quadruplex was a fold-back structure Many other proteins can be shown in vitro to have affinity for such structures, but their functional significance is unclear.

Recently, it has been argued that only the extreme end of vertebrate telomeres form quadruplexes, and a protein proposed to be needed for human telomere unfolding has been characterized Much of the evidence for quadruplex formation in mammalian DNA in vivo is generally circumstantial and to date no high selectivity antibodies for human quadruplex structures are available.

Quadruplex binding ligands typically have rather modest affinity and selectivity even for the small numbers of structures tested, and do not always bind in the manner expected or designed, , New developments in quadruplex-binding ligands show substantially higher affinity and selectivity for the human telomere sequence, and these might be useful for such experiments, with the caveat that the ratio of nonquadruplex DNA to potential quadruplex DNA is very large, and that selectivity also needs to be assessed against protein binding and a wide variety of alternative DNA structures.

The biology of telomeres was summarized in a recent review regarding their need for maintenance, and what happens when the length is not maintained This is called the t-loop mechanism — Although the length of the telomeres is much greater than the persistence length of DNA at physiological salt concentrations, opening of an internal loop within the duplex and forming in essence an intermolecular duplex is, as explained above, energetically unfavorable.

In fact, it was observed that in the absence of a stabilizing factor, the efficiency of t-loop formation was very low; only substantial formation of a loop structure was observed in the presence of the telomere specific protein TRF2 Indeed, the loop model does not formally require strand invasion, only that there is some interaction stabilized by for example protein.

Indeed, it has recently been argued that just the single strand-overhangs form a t-loop stabilized by what amounts to a dimeric G-quadruplex However, this is not in agreement with original models based on large loops that imply circularization of the lengthy double-stranded portion of the telomere.

Although the binding energy for a loop of this size would be small if the DNA were naked, the entropic cost could be quite substantial, as the probability of finding a small number of configurations in which the end is close to a specific part of the telomere is small compared with all the other possible configurations We note that the chromatin rearrangement implies connection of the physical problem to metabolism via ATP hydrolysis and acetylation events.

This also begs the question as to the remarkable length variation of telomeres, even in mammals. It is clear from the foregoing that despite the extensive efforts expended to date, we still have a very limited view of what determines the folding and relative stability of different quadruplex structures, and how fast they form.

Even worse, for the most part the relationship to in vivo formation is even murkier. The latter is a consequence of at least two factors. First is that in vitro experiments concentrate on simple, relatively well-defined systems under conditions that typically do not approximate those found in vivo, as described in part in Discussion section.

Furthermore, it turns out that even the short intramolecular complexes are surprisingly complex, and detailed analysis is hampered by the presence of multiple conformations and slow folding kinetics.

Nevertheless, the basic principles of the energetics of G-quadruplex formation do need to be considered in any biological model, as well as the question as to where the energy comes from.

Thus, the thermodynamics and kinetics of small oligonucleotides have value in that it is possible to examine them in great detail under a wide variety of well-controlled conditions.

This allows one to sample the possible range of behaviors within a realistic range energies and timescales, which ultimately must be relevant to the biological conditions.

It appears that naked G-potential DNA alone has physical properties that are not commensurate with some of the proposed biological functions and thus requires protein binding to provide the energy to manipulate these properties.

It is further notable that some of the proposed functions of such DNA, such as found in promoters, may need to be coupled to unwinding events, and operate far from equilibrium. Under these conditions an understanding of kinetics is essential.

We have argued that the specific ion binding and general electrostatics are a critical component for the energetics, and folding rates of G-quadruplexes, that distinguish them from most other DNA or RNA folds.

However, there are disturbing variations in basic thermodynamic properties that need to be addressed to establish whether these variations arise from experimental artifacts or there are real differences that are sensitive to conditions and small variation in sequence.

To accomplish this goal, we believe that an agreed upon and reliable set of rapid and inexpensive techniques is needed to determine the topology of any quadruplex, and assess its purity. This is likely to come from a combination high-resolution spectroscopic and hydrodynamic techniques, such as NMR and AUC.

First, methods are needed for rapid assessment of the topological structure s that is are present and whether the state is unique and thus can be manipulated by variation of conditions until it is.

This is a prerequisite for subsequent structural and functional analysis, purely as an analytical quality control. Second, it would make it possible to prepare a standardized set of oligomers of known structures to determine whether even simpler methods that exist can make useful distinctions among possible structures database approach.

To this end, we propose that a consortium be established to address major unresolved questions, or at least enlighten the present authors who confess having more questions than answers.

Some of these are listed below. Finally, there is a substantial literature on targeting G-quadruplexes for possible anticancer drug development 18 , 60 , which is beyond the scope of this review.

However, the physical principles apply equally to this area. This work was funded in part by NIH grant No. Funding to pay the open access charge for this article has been waived by NAR. We thank Paula J.

Bates and a reviewer for a critical reading of the article and valuable comments. Your ideas and opinions can help provide a better service for the life sciences research community.

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The Purchase Plan, as amended and restated, will be approved if the votes cast in favor of approval exceed the votes cast against approval. The principal reason for the amendment relating to the number of shares issuable is to allow the Company to continue to make equity awards under the Plan.

There are presently approximately 24, shares of common stock available for granting under the Plan. Principal Features of the Plan. The Board administers the Plan and has the complete discretion to determine when to grant incentive awards; which eligible non-employee directors will receive incentive awards; whether the award will be an option, a stock appreciation right, restricted stock, restricted stock units or stock; and the number of shares to be allocated to each incentive award.

The Board may impose conditions on the exercise of options and stock appreciation rights and on the transfer of restricted stock received under the Plan and may impose such other restrictions and requirements as it may deem appropriate.

The Plan is intended to conform to the provisions of Rule 16b-3 of the Securities Exchange Act of, as amended. Options may be exercised only at such times as specified by the Board.

If the option provides, an optionee exercising an option may pay the purchase price in cash, by delivering shares of common stock, or by delivering an exercise notice together with irrevocable instructions to a broker to promptly deliver to the Company the amount of sale or loan proceeds from the option shares to pay the exercise price.

The Board may award stock appreciation rights under the Plan. When the stock appreciation right is exercisable, the holder may surrender to the Company all or a portion of the unexercised stock appreciation right and receive in exchange an amount equal to the difference between i the fair market value on the date of exercise and ii the fair market value on the date the stock appreciation right was awarded.

Restricted stock units may be granted on the terms and conditions set by the Board with the same limits as for restricted stock. In the case of restricted stock units, no shares are issued at the time of grant.

Rather, upon the lapse of all restrictions, a restricted stock unit entitles a participant to receive shares of common stock or a cash amount equal to the fair market value of a share of common stock on the date the restrictions lapse.

This summary of the principal features of the Plan is qualified by reference to the actual provisions of the Plan, a copy of which is available to any shareholder or director upon written request to the Company.

Options and stock appreciation rights may be transferable by a participant and exercisable by a person other than a participant, but only to the extent specifically provided in the terms of the award.

Federal Income Tax Consequences. A non-employee director does not incur federal income tax when granted a nonstatutory stock option, a stock appreciation right, restricted stock or restricted stock unit.

In general, a non-employee director who has received shares of restricted stock or restricted stock units will include in gross income as compensation income an amount equal to the fair market value of the shares of restricted stock or restricted stock units at the time the restrictions lapse or are removed.

Such amount will be included in income in the tax year in which such event occurs. A non-employee director will incur federal income tax when he or she is awarded a stock grant. In general, a participating director who has received a stock grant will include in gross income as compensation income an amount equal to the fair market value of the shares at the time of grant.

This summary of federal income tax consequences of incentive awards granted under the Plan does not purport to be complete. State, local and foreign income taxes also may be applicable to the transactions described above.

Representatives of KPMG LLP will be present at the Annual Meeting, will have the opportunity to make a statement if they so desire and will be available to respond to appropriate questions.

Independent Auditor Fees and Pre-approval Policies. Audit Fees include fees billed for the audit of the consolidated financial statements, quarterly reviews of unaudited financial statements, accounting advice and services related to registration statements filed with the SEC.

Audit Related Fees include fees billed for audits of employee benefit plans, Sarbanes-Oxley documentation assistance, attestation services related to securitization activities and due diligence assistance on a proposed acquisition.

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If any other business properly comes before the meeting, your proxy may be voted by the persons named in it in such manner as they deem proper. In addition, Section 1.

The notice must be received 1 on or after February 1st and before March 1st of the year in which the meeting will be held, if clause 2 is not applicable, or 2 not less than 90 days before the date of the meeting if the date for that meeting prescribed in the bylaws has been changed by more than 30 days.

A proposal that any shareholder desires to have included in the proxy statement for the annual meeting of shareholders must be received by the Company no later than January 8, The purpose of the committee will be to: In its capacity as a committee of the Board of Directors, be directly responsible for the appointment, compensation, retention and oversight of the work of the independent auditor.

Consider, in consultation with the independent auditor, the internal audit directors and the chief financial officer, the audit scope and plan of the independent auditor and the internal auditors.

Role of the Audit Committee. This charter assigns oversight responsibilities to the audit committee. The independent auditor is responsible for performing an independent audit of the consolidated financial statements in accordance with generally accepted auditing standards.

The members of the audit committee are not acting as experts in accounting or auditing and rely without independent verification on the information provided to them and on the representations made by management and the independent auditor.

The Circuit City Stores, Inc. For the purposes of the Plan the following terms have the stated definitions. Additional terms are defined in the sections below. Amount of Stock Subject to the Plan.

The total number of shares of Common Stock which may be purchased under the Plan shall be 15,,, subject to adjustment as provided in Section Such shares may be newly issued shares that have been authorized but not yet issued or may be shares purchased for Participating Employees on the open market.

All present and future Employees of the Circuit City Companies who have been employed by a Circuit City Company for at least one year are eligible to participate in the Plan, except: If an Employee has one year of service but is excluded from participation in the Plan due to the requirements set forth in i or ii in the preceding paragraph, the Employee will be eligible to participate in the Plan on the first Enrollment Date after he or she is no longer excluded because of such requirements.

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An Employee seeking to participate in the Plan must deliver an Enrollment Form to the Benefits Department so that it is received sufficiently prior to the Enrollment Date to allow processing by the Benefits Department.

The Plan Administrator may establish a submission deadline for Enrollment Forms. Payroll Deductions and Limitations. The Plan Administrator shall have the power to change these percentage limitations.

Changes in Payroll Deductions. A Participating Employee may change the percentage of his or her payroll deductions, subject to the minimum, maximum and allowed increments set forth in Section 6.

To accomplish this, the Participating Employee must submit to the Benefits Department a new Enrollment Form stating the new deduction percentage. The change will be effective as of the first of the next month if the Enrollment Form is received sufficiently prior to the first of the month to allow processing by the Benefits Department.

Deadlines for submission of Enrollment Forms for the purpose of changing payroll deductions may be established by the Plan Administrator. A Participating Employee may also elect to stop making contributions in the manner described in Section On a date as soon as practicable following receipt of the funds, the Plan Service Provider shall arrange for the purchase of Common Stock on the open market.

The Committee shall have the right at any time or from time to time upon written notice to the Plan Service Provider to change the default dividend reinvestment policy for future ESPP Accounts which are established under the Plan.

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The Committee shall make appropriate adjustments in the number of shares of Common Stock which may be purchased under the Plan if there are changes in the Common Stock by reason of stock dividends, stock splits, reverse stock splits, recapitalizations, mergers or consolidations.

A Participating Employee may stop his or her contributions by completing the appropriate section of the Enrollment Form and delivering the form to the Benefits Department.

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FOR the election of the nominees for director named in this proxy statement;. Hardymon joined Textron, Inc. He became Chief Executive Officer in and assumed the title of Chairman in He is a director of Air Products and Chemicals, Inc.

He has been a director of the Company since Prior to his election as Chief Executive Officer, Mr. King was Chief Operating Officer for more than five years. He has been a director of the Company since September She has been a director of the Company since GOINGS, 58, Chairman and Chief Executive Officer of Tupperware Corporation, a direct seller of premium food storage, preparation and serving items as well as premium beauty and skin care products, since He is a director of R.

Reynolds Tobacco Holdings, Inc. He has been a director of the Company since March His present term will expire in She is a director of Rare Hospitality International, Inc.

Paul Travelers Companies, Inc. Her present term will expire in She is a director of VF Corporation. Before joining the faculty at Columbia, Mr. He is a director of Bluefly, Inc.

He is a director of VF Corporation. Salovaara was a partner of Greycliff Partners, a merchant banking firm, from to Less than 1 percent of class, based on the total number of shares of common stock outstanding on April 21, McCollough also is a director of the Company.

Includes shares of common stock that could be acquired through the exercise of stock options within 60 days after April 21, Includes restricted shares of common stock as follows: Dias 45,; and, held by other executive officers.

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Leds serie 922v reset kx to – how dapic para windows

Equity-based awards are generally made on the date of the annual meeting. McCollough did not receive a performance bonus for the fiscal year. Per unit price or other underlying value of transaction computed pursuant to Exchange Act Rule set forth the amount on which the filing fee is calculated and state how it was determined: We have argued that the specific ion binding and general electrostatics are a critical component for the energetics, and folding rates of G-quadruplexes, that distinguish them from most other DNA or RNA folds. Crystal structure of parallel quadruplexes from human telomeric DNA.

Variation of these latter parameters can provide information about additional molecular properties of the system, and indirectly about the forces involved in stability. Abstract In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. These changes provide a convenient window for monitoring denaturation, and entry into the thermodynamics of the denaturation process. Extended phase I study of AS in renal and non-small cell lung cancers.

Frequently, the observed melting curves are not so simple, in part because the optical parameters [e. Quadruplexes have also been shown to have antiviral activity and have been demonstrated to be effective against HIV-1 in vitro 18 , FOR the election of the nominees for director named in this proxy statement;.

See…

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Segundos canal to 922v kx reset dapic – how win bit

Sugimoto, have used crowding conditions in vitro to determine the influence on conformation and stability. To withhold authority to vote for any indicated nominee, write the number s of the nominee s in the box provided to the right. Crystal structure of parallel quadruplexes from human telomeric DNA. See…

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