Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes

Cations are known to mediate diverse interactions in nucleic acids duplexes but they are critical in the arrangement of four-stranded structures. Here, we use all-atom molecular dynamics simulations with explicit solvent to analyse the mechanical unfolding of representative intramolecular G-quadruplex structures: a parallel, a hybrid and an antiparallel DNA and a parallel RNA, in the presence of stabilising cations. We confirm the stability of these conformations in the presence of central ions and observe distortions from the tetrad topology in their absence. Force-induced unfolding dynamics is then investigated. We show that the unfolding events in the force-extension curves are concomitant to the loss of coordination between the central ions and the guanines of the G-quadruplex. We found lower ruptures forces for the parallel configuration with respect to the antiparallel one, while the behaviour of the force pattern of the parallel RNA appears similar to the parallel DNA. We anticipate that our results will be essential to interpret the fine structure rupture profiles in stretching assays at high resolution and will shed light on the mechanochemical activity of G-quadruplex-binding machinery.     

A sequence-independent study of the influence of short loop lengths on the stability and topology of intramolecular DNA G-quadruplexes

G-Rich sequences found within biologically important regions of the genome have been shown to form intramolecular G-quadruplexes with varied loop lengths and sequences. Many of these quadruplexes will be distinguishable from each other on the basis of their thermodynamic stabilities and folded conformations. It has been proposed that loop lengths can strongly influence the topology and stability of intramolecular G-quadruplexes. Previous studies have been limited to the analysis of quadruplex sequences with particular loop sequences, making it difficult to make generalizations. Here, we describe an original study that aimed to elucidate the effect of loop length on the biophysical properties of G-quadruplexes in a sequence-independent context. We employed UV melting and circular dichroism spectroscopy to examine and compare the properties of 21 DNA quadruplex libraries, each comprising partially randomized loop sequences with lengths ranging from one to three nucleotides. Our work supports a number of general predictions that can be made solely on the basis of loop lengths. In particular, the results emphasize the strong influence of single-nucleotide loops on quadruplex properties. This study provides a predictive framework that may help identify or classify biologically relevant G-quadruplex-forming sequences.     

Tandem GA residues on opposite sides of the loop in molecular beacon-like DNA hairpins compact the loop and increase hairpin stability

The free solution electrophoretic mobilities and thermal stabilities of hairpins formed by two complementary 26-nucleotide oligomers have been measured by capillary electrophoresis. The oligomers are predicted to form molecular beacon-like hairpins with 5 bp stems and 16 nucleotides in the loop. One hairpin, called hairpin2 (hp2), migrates with a relatively fast free solution mobility and exhibits melting temperatures that are reasonably well predicted by the popular structure-prediction program Mfold. Its complement, called hairpin1 (hp1), migrates with a slower free solution mobility and forms a stable hairpin only in solutions containing ≥200 mM Na(+). The melting temperatures observed for hp1 are ~18 °C lower than those observed for hp2 and ~20 °C lower than those predicted by Mfold. The greater thermal stability of hp2 is due to the presence of tandem GA residues on opposite sides of the loop. If the corresponding TC residues in the hp1 loop are replaced by tandem GA residues, the melting temperatures of the modified hairpin are close to those observed for hp2. Eliminating the tandem GA residues in the hp2 loop significantly decreases the thermal stability of hp2. If the loops are replaced by a loop of 16 thymine residues, the free solution mobilities and thermal stabilities of the T-loop hairpin are equal to those observed for hp1. Hence, the loop of hp1 appears to be relatively unstructured, with few base-base stacking interactions. Interactions between tandem GA residues on opposite sides of the hp2 loop appear to compact the loop and increase hairpin stability.     

The stability of intramolecular DNA G-quadruplexes compared with other macromolecules

DNA quadruplexes are often conceived as very stable structures. However, most of the free energy of stabilization derives from specific ion binding via inner sphere coordination of the GO6 of the guanine residues comprising the basic quartet. When compared with other nucleic acid structures such as DNA or RNA duplexes and hairpins, or proteins of the same number of atoms, metal-coordinated intramolecular quadruplexes are found to be of comparable or lower thermodynamic stability under similar solution conditions. Furthermore, intramolecular quadruplexes are actually less stable kinetically, than DNA duplexes or hairpins of the same size.     

Predictive modelling of topology and loop variations in dimeric DNA quadruplex structures

We have used a combination of simulated annealing (SA), molecular dynamics (MD) and locally enhanced sampling (LES) methods in order to predict the favourable topologies and loop conformations of dimeric DNA quadruplexes with T2 or T3 loops. This follows on from our previous MD simulation studies on the influence of loop lengths on the topology of intramolecular quadruplex structures [P. Hazel et al. (2004) J. Am. Chem. Soc., 126, 16405–16415], which provided results consistent with biophysical data. The recent crystal structures of d(G4T3G4)2 and d(G4BrUT2G4) (P. Hazel et al. (2006) J. Am. Chem. Soc., in press) and the NMR-determined topology of d(TG4T2G4T)2 [A.T. Phan et al. (2004) J. Mol. Biol., 338, 93–102] have been used in the present study for comparison with simulation results. These together with MM-PBSA free-energy calculations indicate that lateral T3 loops are favoured over diagonal loops, in accordance with the experimental structures; however, distinct loop conformations have been predicted to be favoured compared to those found experimentally. Several lateral and diagonal loop conformations have been found to be similar in energy. The simulations suggest an explanation for the distinct patterns of observed dimer topology for sequences with T3 and T2 loops, which depend on the loop lengths, rather than only on G-quartet stability.     

Effect of loop sequence and size on DNA aptamer stability

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.     

Studying the effect of crowding and dehydration on DNA G-quadruplexes

Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to 40%) of the cellular volume, with a total concentration in the range 300-400mg/ml. Recently, the effect of crowding/dehydrating agents on the DNA G-quadruplexes has become a subject of an increasing interest. Crowding and/or dehydrating agents have been used to simulate how G-quadruplexes behave under cell-mimicking conditions characterized by a large excluded volume and a lower water activity. Indeed, the presence of both steric crowding and a lower water activity can affect G-quadruplex stability, their folding/unfolding kinetics, as well as their binding processes with proteins or small ligands. Many of these effects can be explored experimentally by measuring the dependence of the conformational stability, isomerisation kinetics and equilibria on the concentration of cosolutes which do not interact with the molecules (G-quadruplexes) under investigation. Spectroscopic methodologies, like circular dichroism, UV and fluorescence, have been widely employed to study G-quadruplexes in dilute solution. Here we focus on some aspects that need to be taken into account when employing such techniques in the presence of large amount of a cosolute. Additionally, we discuss possible problems/artifacts that arise in setting experiments in presence of these commonly employed cosolutes and in interpreting the results.     

A Monte-Carlo estimate of DNA loop formation

The ends of rather short double-helical DNA segments (approximately two persistence lengths) can be enzymatically joined to form closed circles. Such covalent closure into circles is a measure of the likelihood of the two ends of the DNA coming into close contact. There is a length of DNA for which loop formation is most likely to occur. We have determined the chain-length dependence of loop formation for stiff chains using computer-generated chains of cylinders. The distribution from which the values for the angles between cylinders were chosen relates the chain parameters to a given chain persistence. Our results are compared with those of other theories, including a statistical wormlike chain model, and with the experimental measurements for ring closure of DNA restriction fragments.     

The formation pathway of tetramolecular G-quadruplexes

Oligonucleotides containing guanosine stretches associate into tetrameric structures stabilized by monovalent ions. In order to describe the sequence of reactions leading to association of four identical strands, we measured by NMR the formation and dissociation rates of (TGnT)₄ quadruplexes (n = 3–6), their dissociation constants and the reaction orders for quadruplex formation. The quadruplex formation rates increase with the salt concentration but weakly depend on the nature (K⁺, Na⁺ or Li⁺) of the counter ions. The activation energies for quadruplex formation are negative. The quadruplex lifetimes strongly increase with the G-tract length and are much more longer in K⁺ solution than in Na⁺ or Li⁺ solutions. The reaction order for quadruplex formation is 3 in 0.125 M KCl and 4 in LiCl solutions. The kinetics measurements suggest that quadruplex formation proceeds step by step via sequential strand association into duplex and triplex intermediate species. Triplex formation is rate limiting in 0.125 M KCl solution. In LiCl, each step of the association process depends on the strand concentration. Parallel reactions to formation of the fully matched canonical quadruplex may result in kinetically trapped mismatched quadruplexes making the canonical quadruplex practically inaccessible in particular at low temperature in KCl solution.     

Influence of dangling thymidine residues on the stability and structure of two DNA duplexes

We have employed temperature-dependent UV spectroscopy, circular dichroism (CD), 400-MHz proton nuclear magnetic resonance (NMR), and computer modeling to characterize both structurally and thermodynamically the influence of unpaired, dangling thymidine residues (T) on the thermal stability and melting behavior of two DNA core duplexes. The specific DNA double helices that we have investigated in this work are core duplexes [d(GC)3]2 (I) and [d(CG)3]2 (IV), 3' dangling T derivatives [d[(GC)3TT]]2 (II) and [d[(CG)3TT]]2 (V), and 5' dangling T derivatives [d[TT(GC)3]]2 (III) and [d[TT(CG)3]]2 (VI). Our experimental data allow us to reach the following conclusions: (1) For both core duplexes (I and IV), the addition of dangling T residues on either the 5' or 3' end causes an increase in the optical melting temperature tm. (2) For both core duplexes, 5' dangling T residues induce a greater increase in the optical tm's than 3' dangling T residues. (3) For both cores duplexes, the increase in tm induced by the addition of dangling T residues is enthalpic in origin, with 5' dangling T residues inducing a greater increase in the van't Hoff transition enthalpy than 3' dangling T's. (4) Dangling T residues cause downfield shifts in all of the nonexchangeable aromatic protons of the [d(GC)3]2 core duplex (I), with the 5' T residues inducing the largest shifts. For the most part, this trend does not hold with the [d(CG)3]2 core duplex (IV). (5) For both core duplexes, the addition of dangling T residues causes an increase in the NMR tm's of almost all the nonexchangeable aromatic protons of the core duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.     

Promoting the formation of tetramolecular G-quadruplexes under freezing condition

In the study, we demonstrated that freezing was able to facilitate the short DNA strand T(4)G(4) to self-assemble into a tetramolecular G-quadruplex with a low DNA concentration in the absence of any ligand, providing a fast approach to configure intermolecular G-quadruplexes. The fast formation of intermolecular G-quadruplexes may offer a convenient tool.     

Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.     

Single molecule conformational analysis of DNA G-quadruplexes

Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.     

Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg (2+)] (0.5)/[Mon (+)] is less than 0.22 M (-1/2), monovalent ions (K (+), Na (+)) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg (2+) ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.