Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G(4)T(4)G(4) fragment

Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G(4)T(4)G(4), which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G(4)T(4)G(4) quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G(4)T(4)G(3)g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions ( approximately 0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na(+) solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G(4)T(4)G(4) quadruplex in K(+) solutions. Substitutions near the 3'end of the molecule affected folding more than substitutions near the 5'end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 797-806, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

The cohering telomeres of Oxytricha.

We have studied the process by which purified Oxytricha macronuclear DNA associates with itself to form large aggregates. The various macronuclear DNA molecules all have the same terminal or telomeric DNA sequences that are shown below. 5' C4A4C4A4C4--mean length----G4T4G4T4G4T4G4T4G4 G4T4G4T4G4T4G4T4G4-----2.4 kb------C4A4C4A4C4. When incubated at high concentrations, these telomeric sequences cohere with one another to form an unusual structure--one that is quite different from any DNA structure so far described. The evidence for this is the following: 1) These sequences cohere albeit slowly, in the presence of relatively high concentrations of Na+, and no other cation tested. This contrasts with the rapid coherence of complementary single-chain terminals of normal DNA (sticky ends) which occurs in the presence of any cation tested. 2) If the cohered form is transferred into buffers containing a special cation, K+, it becomes much more resistant to dissociation by heating. We estimate that K+ increases the thermal stability by 25 degrees or more. The only precedent known (to us) for a cation-specific stabilization is that seen in the quadruplex structure formed by poly I. The thermal stability of double helical macronuclear DNA depends on the cation concentration, but not the cation type. Limited treatment with specific nucleases show that the 3' and 5'-ended strands are essential for the formation of the cohering structure. Once in the cohered form, the telomeric sequences are protected from the action of nucleases. Coherence is inhibited by specific, but not by non-specific, synthetic oligomers, and by short telomeric fragments with or without their terminal single chains. We conclude that the coherence occurs by the formation of a novel condensed structure that involves the terminal nucleotides in three or four chains.     

Guanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromo-guanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5' end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.     

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.     

Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.     

The insertion of two 8-methyl-2'-deoxyguanosine residues in tetramolecular quadruplex structures: trying to orientate the strands

In this article, we report a structural study, based on NMR and CD spectroscopies, and molecular modelling of all possible d(TG(3)T) and d(TG(4)T) analogues containing two 8-methyl-2'-deoxyguanosine residues (M). Particularly, the potential ability of these modified residues to orientate the strands and then to affect the folding topology of tetramolecular quadruplex structures has been investigated. Oligodeoxynucleotides (ODNs) TMMGT (T12) and TMMGGT (F12) form parallel tetramolecular quadruplexes, characterized by an all-syn M-tetrad at the 5'-side stacked to all-anti M- and G-tetrads. ODNs TMGMT (T13) and TMGGMT (F14) form parallel tetramolecular quadruplexes, in which an all-anti G core is sandwiched between two all-syn M-tetrads at the 5'- and the 3'-side. Notably, the quadruplex formed by T13 corresponds to an unprecedented structure in which the syn residues exceed in number the anti ones. Conversely, ODN TGMGMT (F24) adopts a parallel arrangement in which all-anti G-tetrads alternate with all-syn M-tetrads. Most importantly, all data strongly suggest that ODN TMGMGT (F13) forms an unprecedented anti-parallel tetramolecular quadruplex in which G and M residues adopt anti and syn glycosidic conformations, respectively. This article opens up new understandings and perspectives about the intricate relationship between the quadruplex strands orientation and the glycosidic conformation of the residues.     

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.     

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.     

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3‐methyl isoxanthopterin

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3‐methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI‐based QPA is a useful assay for DNA amplification and detection. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 583–590, 2014.     

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG₃T) and its modified analogs containing a 5′-5′ or 3′-3′ inversion of polarity sites, namely d(3′TG5′-5′G₂T3′), d(3′T5′-5′G₃T3′) and d(5′TG3′-3′G₂T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 5′-3′ backbone. Exchange of ¹⁵ ions between G-quadruplex and bulk solution is faster at the 3′-end in comparison to the 5′-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG₃T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures.     

Site-resolved energetics in DNA triple helices containing G*TA and T*CG triads

Recognition of specific sites in double-helical DNA by triplex-forming oligonucleotides has been limited until recently to sites containing homopurine-homopyrimidine sequences. G*TA and T*CG triads, in which TA and CG base pairs are specifically recognized by guanine or by thymine, have now extended this recognition code to DNA target sites of mixed base sequences. In the present work, we have obtained a characterization of the stabilities of G*TA and T*CG triads, and of the effects of these triads upon canonical triads, in triple-helical DNA. The three DNA triplexes investigated are formed by the folding of the 31-mers d(GAAXAGGT(5)CCTYTTCT(5)CTTZTCC) with X = G, T, or C, Y = C, A, or G, and Z = C, G, or T. We have measured the exchange rates of imino protons in each triad of the three triplexes using nuclear magnetic resonance spectroscopy. The exchange rates are used to map the local free energy of structural stabilization in each triplex. The results indicate that the stability of Watson-Crick base pairs in the G*TA and T*CG triads is comparable to that of Watson-Crick base pairs in canonical triads. The presence of G*TA and T*CG triads, however, destabilizes neighboring canonical triads, two or three positions removed from the G*TA/T*CG site. Moreover, the long-range destabilizing effects induced by the T*CG triad are larger than those induced by the G*TA triad. These findings reveal the molecular basis for the lower overall stability of G*TA- and T*CG-containing triplexes.     

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.     

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.     

Intramolecular and intermolecular guanine quadruplexes of DNA in aqueous salt and ethanol solutions

DNA guanine quadruplexes are all based on stacks of guanine tetrads, but they can be of many types differing by mutual strand orientation, topology, position and structure of loops, and the number of DNA molecules constituting their structure. Here we have studied a series of nine DNA fragments (G(3)Xn)(3)G(3), where X = A, C or T, and n = 1, 2 or 3, to find how the particular bases and their numbers enable folding of the molecule into quadruplex and what type of quadruplex is formed. We show that any single base between G(3) blocks gives rise to only four-molecular parallel-stranded quadruplexes in water solutions. In contrast to previous models, even two Ts in potential loops lead to tetramolecular parallel quadruplexes and only three consecutive Ts lead to an intramolecular quadruplex, which is antiparallel. Adenines make the DNA less prone to quadruplex formation. (G(3)A(2))(3)G(3) folds into an intramolecular antiparallel quadruplex. The same is true with (G(3)A(3))(3)G(3) but only in KCl. In NaCl or LiCl, (G(3)A(3))(3)G(3) prefers to generate homoduplexes. Cytosine still more interferes with the quadruplex, which only is generated by (G(3)C)(3)G(3), whereas (G(3)C(2))(3)G(3) and (G(3)C(3))(3)G(3) generate hairpins and/or homoduplexes. Ethanol is a more potent DNA guanine quadruplex inducer than are ions in water solutions. It promotes intramolecular folding and parallel orientation of quadruplex strands, which rather corresponds to quadruplex structures observed in crystals.     

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.     

NMR observation of T-tetrads in a parallel stranded DNA quadruplex formed by Saccharomyces cerevisiae telomere repeats.

We report here the NMR structure of the DNA sequence d-TGGTGGC containing two repeats of Saccharomyces cerevisiae telomere DNA which is unique in that it has a single thymine in the repeat sequence and the number of Gs can vary from one to three. The structure is a novel quadruplex incor-porating T-tetrads formed by symmetrical pairing of four Ts via O4-H3 H-bonds in a plane. This is in contrast to the previous results on other telomeric sequences which contained more than one T in the repeat sequences and they were seen mostly in the flexible regions of the structures. We observed that the T4-tetrad was nicely accommodated in the center of the G-quadruplex, but it caused a small underwinding of the right handed helix. The T tetrad stacked well on the adjacent G3-tetrad, but poorly on the G5 tetrad. Likewise, T1 also formed a stable T-tetrad at the 5' end of the quadruplex. To our knowledge, this is the first report of T-tetrad formation in DNA structures. These observations are of significance from the points of view of both structural diversity and specific recognitions.     

Structural polymorphism of telomeric DNA regulated by pH and divalent cation

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1:1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex structure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.     

Structural properties of the [d(G3T4G3)]2 quadruplex: evidence for sequential syn-syn deoxyguanosines.

Two-dimensional 1H NMR studies on the dimeric hairpin quadruplex formed by d(G3T4G3) in the presence of either NaCl or KCl are presented. In the presence of either salt, the quadruplex structure is characterized by half the guanine nucleosides in the syn conformation about the glycosidic bond, the other half in the anti conformation, as reported for other similar sequences. However, 1H NOESY and 1H-31P heteronuclear correlation experiments demonstrate that the deoxyguanosines do not strictly alternate between syn and anti along individual strands. Thus we find the following sequences with regard to glycosidic bond conformation: 5'-G1SG2SG3AT4AT5A-T6AT7AG8SG9AG10A-3' and 5'-G11SG12AG13AT14AT1 5AT16AT17AG18SG19SG20A-3', where S and A denote syn and anti, respectively. This represents the first experimental evidence for a nucleic acid structure containing two sequential nucleosides in the syn conformation. The stacking interactions of the resulting quadruplex quartets and their component bases have been evaluated using unrestrained molecular dynamics calculations and energy component analysis. These calculations suggest that the sequential syn-syn/anti-anti and syn-anti quartet stacks are almost equal in energy, whereas the anti-syn stack, which is not present in our structure, is energetically less favorable by about 4 kcal/mol. Possible reasons for this energy difference and its implications for the stability of quadruplex structures are discussed.     

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(3))). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation.     

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.