Influence of loop size on the stability of intramolecular DNA quadruplexes

We have determined the stability of intramolecular DNA quadruplexes in which the four G₃-tracts are connected by non-nucleosidic linkers containing propanediol, octanediol or hexaethylene glycol, replacing the TTA loops in the human telomeric repeat sequence. We find that these sequences all fold to form intramolecular complexes, which are stabilized by lithium < sodium < potassium. Quadruplex stability increases in the order propanediol < hexaethylene glycol < octanediol. The shallower shape of the melting profile with propanediol linkers and its lower dependency on potassium concentration suggests that this complex contains fewer stacks of G-quartets. The sequence with octanediol linkers displays a biphasic melting profile, suggesting that it can adopt more than one stable structure. All these complexes display melting temperatures above 310 K in the presence of 10 mM lithium, without added potassium, in contrast to the telomeric repeat sequence. These complexes also fold much faster than the telomeric repeat and there is little or no hysteresis between their melting and annealing profiles. In contrast, the human telomeric repeat sequence and a complex containing two hexaethylene glycol groups in each loop, are less stable and fold more slowly. The melting and annealing profiles for the latter sequence show significant differences, even when heated at 0.2°C min^–1. CD spectra for the oligonucleotides containing non-nucleosidic linkers show positive maxima at 264 nm, with negative minima ~244 nm, which are characteristic of parallel quadruplex structures. These results show that the structure and stability of intramolecular quadruplexes is profoundly influenced by the length and composition of the loops.     

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.     

Kinetic stability of intermolecular DNA quadruplexes

Fluorescently labeled oligodeoxyribonucleotides containing a single tract of four successive guanines have been used to study the thermodynamic and kinetic properties of short intermolecular DNA quadruplexes. When these assemble to form intermolecular quadruplexes the fluorophores are in close proximity and the fluorescence is quenched. On raising the temperature these complexes dissociate and there is a large increase in fluorescence. These complexes are exceptionally stable in potassium-containing buffers, and possess Tm values that are too high to measure. Tm values were determined in sodium-containing buffers for which the rate of reannealing is extremely slow; the melting profiles are effectively irreversible, and the apparent melting temperatures are dependent on the rates of heating. The dissociation kinetics of these complexes was estimated by rapidly increasing the temperature and following the time-dependent changes in fluorescence. From these data we have estimated the half-lives of these quadruplexes at 37 degrees C. Addition of a T to the unlabeled end of the oligonucleotide increases quadruplex stability. In contrast, addition of a T between the fluorophore and the oligonucleotide leads to a decrease in stability.     

A sequence-independent analysis of the loop length dependence of intramolecular RNA G-quadruplex stability and topology

G-Quadruplexes are noncanonical nucleic acid secondary structures based on guanine association that are readily adopted by G-rich RNA and DNA sequences. Naturally occurring genomic G-quadruplex-forming sequences have functional roles in biology that are mediated through structure. To appreciate how this is achieved, an understanding of the likelihood of G-quadruplex formation and the structural features of the folded species under a defined set of conditions is informative. We previously systematically investigated the thermodynamic stability and folding topology of DNA G-quadruplexes and found a strong dependence of these properties on loop length and loop arrangement [Bugaut, A., and Balasubramanian, S. (2008) Biochemistry 47, 689-697]. Here we report on a complementary analysis of RNA G-quadruplexes using UV melting and circular dichroism spectroscopy that also serves as a comparison to the equivalent DNA G-quadruplex-forming sequences. We found that the thermodynamic stability of G-quadruplex RNA can be modulated by loop length while the overall structure is largely unaffected. The systematic design of our study also revealed subtle loop length dependencies in RNA G-quadruplex structure.     

A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes

Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5–18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (ΔG₃₇) ranging from −1.7 to −15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex–duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.     

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.     

Effect of length,topology, and concentration on the microviscosity and microheterogeneity of DNA solutions

The viscoelastic behavior of chromosomal DNA, which is heterogeneously distributed within the nucleus, may influence the diffusion of nuclear organelles and proteins. To identify some of the parameters that affect DNA viscoelasticity, we use the high-throughput method of multiple-particle nanotracking to measure the microviscosity and degree of heterogeneity of solutions of chromosomal DNA, linear DNA, and circular double-stranded DNA over a wide range of concentrations and lengths. The thermally excited displacements of multiple fluorescent microspheres imbedded in DNA solutions are monitored with 5nm spatial resolution and 30Hz temporal resolution, from which mean-squared displacement (MSD) and viscosity distributions are generated. For all probed DNA solutions but the most concentrated solution of the longest molecules, the ensemble-averaged MSD increases linearly with time at all probed time scales, a signature of viscous transport. The associated mean viscosity of the DNA solutions increases slowly with concentration for circular DNA and more rapidly for linear DNA, but more slowly than predicted by theory. The heterogeneity of the DNA solutions is assessed by computing the relative contributions of the 10%, 25%, and 50% highest values of MSD and viscosity to the ensemble-averaged MSD and viscosity. For both linear DNA and circular DNA, these contributions are much larger than observed in homogeneous liquids such as glycerol. The microheterogeneity of the linear DNA solutions increases with concentration more significantly for linear DNA than circular DNA. These in vitro results suggest that the topology, local concentration, and length of DNA influence the microrheology and microheterogeneity of the DNA within the nucleus.     

An FTIR and CD study of the structural effects of G-tract length and sequence context on DNA conformation in solution

Fourier transform infrared (FTIR) and CD spectroscopy have been used to investigate the structural effects of G-tract length and flanking sequence on the conformation of DNA G-tracts in aqueous solution. Particularly, a possible predisposition for A-form features has been probed, since this may be important for protein-DNA interactions. Five different G-tract-containing DNA duplexes have been studied: d[CATGGCCATG](2), d[CATGGGCCCATG](2), d[CATGGGGCCCCATG](2,) d[AGGGGCCCCT](2), and d[TGGGGCCCCA](2). In addition, a DNA duplex lacking a G-tract center was probed (d[CATATGCATATG](2)). The CD and FTIR results show that the G-tract-containing sequences are all in a dominating B-DNA conformation in solution. However, certain spectral variations reflect structural effects of sequence context and G-tract length. CD spectra and FTIR results in the 1800-1500 cm(-1) region show that the base-stacking pattern is greatly affected by the sequence context. The FTIR backbone 1250-1000 cm(-1) region shows the antisymmetric non-bridging phosphate vibration around 1225 cm(-1) in all sequences, demonstrating the overall B-conformation of the backbone. The FTIR sugar 900-800 cm(-1) region shows variable contributions of two bands around 865 cm(-1) and 840 cm(-1), reflecting the N and S-type of sugar pucker. The relative intensities of the 865 cm(-1) and 840 cm(-1) bands have been proposed in the literature to quantitatively yield the contribution of N and S-type of sugar pucker, respectively. This correlation is supported by the present study. Furthermore, the contributions of N-type sugar in the DNA sequences studied indicate structural propensities that agree with trends in reported crystal structures of the same sequences: (1) d[CATGGCCATG](2), for which FTIR shows the lowest contribution of N-type sugar puckering in solution, crystallizes in a B-like conformation; (2) d[AGGGGCCCCT](2), with the highest degree of N-type sugar puckering of all the sequences studied, crystallizes in an A-like conformation; (3) d[CATGGGCCCATG](2), with an N-type contribution intermediate between that of d[CATGGCCATG](2) and d[AGGGGCCCCT](2), crystallizes in an A/B intermediate conformation.     

Inosine substitutions demonstrate that intramolecular DNA quadruplexes adopt different conformations in the presence of sodium and potassium

We have examined the stability of fluorescently-labelled oligonucleotides that are based on the human telomeric repeat [(GGGTTA)(3)GGG], in which one of the guanines in turn is substituted with inosine. We show that the relative stability of the substitutions is different in the presence of sodium and potassium. The data for potassium suggest a parallel arrangement of the strands, while the sodium form is mixed parallel and antiparallel.     

Sequence-dependent stability of intramolecular DNA triple helices

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

Stability of intramolecular quadruplexes: sequence effects in the central loop

Hundreds of thousands of putative quadruplex sequences have been found in the human genome. It is important to understand the rules that govern the stability of these intramolecular structures. In this report, we analysed sequence effects in a 3-base-long central loop, keeping the rest of the quadruplex unchanged. A first series of 36 different sequences were compared; they correspond to the general formula GGGTTTGGGHNHGGGTTTGGG. One clear rule emerged from the comparison of all sequence motifs: the presence of an adenine at the first position of the loop was significantly detrimental to stability. In contrast, adenines have no detrimental effect when present at the second or third position of the loop. Cytosines may either have a stabilizing or destabilizing effect depending on their position. In general, the correlation between the T_m or ΔG° in sodium and potassium was weak. To determine if these sequence effects could be generalized to different quadruplexes, specific loops were tested in different sequence contexts. Analysis of 26 extra sequences confirmed the general destabilizing effect of adenine as the first base of the loop(s). Finally, analysis of some of the sequences by microcalorimetry (DSC) confirmed the differences found between the sequence motifs.     

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.     

Effect of the incorporation of 2'-deoxy-8-(hydroxyl)adenosine on the stability of quadruplexes formed by modified human telomeric DNA

Differential scanning calorimetry (DSC) and circular dichroism (CD) techniques were used to investigate the physico-chemical properties of the quadruplexes formed by the two different truncations of human telomeric sequence d(TAGGGT) and d(AGGGT), where the adenines were substituted by 2'-deoxy-8-(hydroxyl)adenosine (A --> A OH). CD spectra show that the modified sequences are able to form parallel-stranded quadruplex structure. Analysis of the thermodynamic parameters reveals that the introduction of the modified adenine affects in different way the thermal stability of the [d(TAGGGT)]4 and [d(AGGGT)]4 quadruplexes.     

Effect of DNA topology on plasmid DNA repair in vivo

Escherichia coli nucleotide excision repair (NER) is responsible for removing bulky DNA adducts by dual incisions of the UvrABC endonuclease. Although the activity of the UvrAB complex which can induce DNA conformational change is employed in NER, the involvement of DNA topology and DNA topoisomerases remains unclear. We examined the effect of topoisomerase inhibitions on a NER in vivo system. The repair analysis of intracellular plasmid revealed that the DNA damage on positive supercoils generated by gyrase inhibition remained unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants. These results suggest that DNA topology affects the NER process and the removal of positive supercoils by gyrase is vital for the efficiency of the E. coli NER system.     

Effect of chain length on coiled-coil stability: decreasing stability with increasing chain length

The de novo design and biophysical characterization of three series of two-stranded alpha-helical coiled coils with different chain lengths are described. Our goal was to examine how increasing chain length would affect protein folding and stability when one or more heptad repeat(s) of K-A-E-A-L-E-G (gabcdef) was inserted into the central region of different coiled-coil host proteins. This heptad was designed to maintain the continuous 3-4 hydrophobic repeat of the coiled-coil host and introduce an Ala and Leu residue in the hydrophobic core at the a and d position, respectively, and a pair of stabilizing interchain ionic i to i' + 5 (g to e') interactions per heptad inserted. The secondary structures of the three series of disulfide-bridged polypeptides were studied by CD spectroscopy and their stabilities determined by chemical and thermal denaturation. The results showed that successive insertions of this heptad systematically decreased the stability of all the coiled coils studied regardless of the overall initial stability of the host coiled coil. These observations are in contrast to the generally accepted implication that the folding and stability of coiled coils are enhanced with increasing chain length. Our results imply that, in these examples where an Ala and Leu hydrophobic residue were introduced into the coiled-coil core per inserted heptad, there was still insufficient stability to overcome unfavorable entropy associated with chain length extension, even though the inserted heptad contained the most stabilizing hydrophobic residue (Leu) at position d and stabilizing ionic attractions.     

Effect of cross-linker length on the stability of hemoglobin

A series of cross-linking reagents with 4 to 7 carbons have been synthesized and used to modify human hemoglobin. The product yields and biochemical properties of these cross-linked hemoglobins are compared to those made with both longer and shorter cross-linkers. Several trends become apparent. The yields decrease as the cross-linker becomes longer, which correlates well with molecular dynamics studies of reagent binding pathways presented here. The autooxidation rates increase while thermal stability decreases with longer reagents. Cross-linking under deoxy conditions also increases autooxidation rates, but the effect is less than that of increased cross-linker length. The results suggest that shorter reagents may provide better-stabilized tetramers for the construction of more complex hemoglobin-based oxygen carriers.     

Explaining the varied glycosidic conformational, G-tract length and sequence preferences for anti-parallel G-quadruplexes

Guanine-rich DNA sequences tend to form four-stranded G-quadruplex structures. Characteristic glycosidic conformational patterns along the G-strands, such as the 5'-syn-anti-syn-anti pattern observed with the Oxytricha nova telomeric G-quadruplexes, have been well documented. However, an explanation for these featured glycosidic patterns has not emerged. This work presents MD simulation and free energetic analyses for simplified two-quartet [d(GG)](4) models and suggests that the four base pair step patterns show quite different relative stabilities: syn-anti > anti-anti > anti-syn > syn-syn. This suggests the following rule: when folding, anti-parallel G-quadruplexes tend to maximize the number of syn-anti steps and avoid the unfavorable anti-syn and syn-syn steps. This rule is consistent with most of the anti-parallel G-quadruplex structures in the Protein Databank (PDB). Structural polymorphisms of G-quadruplexes relate to these glycosidic conformational patterns and the lengths of the G-tracts. The folding topologies of G2- and G4-tracts are not very polymorphic because each strand tends to populate the stable syn-anti repeat. G3-tracts, on the other hand, cannot present this repeating pattern on each G-tract. This leads to smaller energy differences between different geometries and helps explain the extreme structural polymorphism of the human telomeric G-quadruplexes.     

Studies on genomic DNA topology and stability in brain regions of Parkinson's disease

DNA damage has been postulated as a mechanism of neuronal death in Parkinson's disease (PD). In the present study, genomic DNA was isolated from eight brain regions (frontal, temporal, and occipital cortex, hippocampus, caudate/putamen, thalamus, cerebellum, and midbrain) from five neuropathologically confirmed cases of Parkinson's disease and six control brains and analyzed for the presence of single and double strand breaks, melting temperature, EtBr intercalation, DNAse digestion pattern, and DNA conformations. The results showed that DNA from midbrain in PD accumulated significantly higher number of strand breaks than age-matched controls. Caudate nucleus/putamen, thalamus, and hippocampus also showed more DNA fragmentation compared to control brains. Circular dichroism studies showed that DNA conformation was altered with imprecise base stacking in midbrain, caudate nucleus/putamen, thalamus, and hippocampus in PD. However, DNA from frontal, temporal, and occipital cortex, and cerebellum was not affected significantly in PD group as compared to controls. This study provides a comprehensive database on stability, damage, and conformations of DNA in different regions in brains of PD patients.     

Effect of chain length on the stability of lecithin bilayers

The shift reagent NaCl₃ was added to vesicles of synthetic, saturated (DiC₁₀-C₁₆) lecithins and egg lecithin and the accessibility of the N(CH₃)₃ groups to Na³⁺ ions was studied by NMR. Long chain lecithins, e.g. dipalmitoyl and egg lecithin form bilayers “stable” on the time scale of our experiments and practically impermeable to cations. Short chain lecithins on the other hand form short-lived vesicles surrounded by unstable bilayers which are not effective cation barriers. Ion transport across the latter lecithin bilayers may involve, besides passive diffusion, collision-induced transient rupture and resealing of bilayers coupled with ion movement.     

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.