Sequence effects of single base loops in intramolecular quadruplex DNA

We have examined the properties of intramolecular G-quadruplexes in which the G3 tracts are separated by single base loops. The most stable complex contained 1',2'-dideoxyribose in all three loops, while loops containing T and C were slightly less stable (by about 2 degrees C). Quadruplexes containing loops with single A residues were less stable by 8 degrees C for each T to A substitution. These folded sequences display similar CD spectra, which are consistent with the formation of parallel stranded complexes with double-chain reversal loops. These results demonstrate that loop sequence, and not just length, affects quadruplex stability.     

An in silico study of the differential effect of oxidation on two biologically relevant g-quadruplexes: possible implications in oncogene expression

G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential "hot-spots" for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.     

Determinants of DNA quadruplex structural type: sequence and potassium binding

There are DNA sequences which adopt the same quadruplex structural type in the presence of sodium as in the presence of sodium and potassium. There are also sequences that appear to have a requirement for the presence of potassium for the adoption of a particular quadruplex structural type. Information about the basis for these potassium effects has been obtained by examining the structures of a set of DNAs with differing numbers of loop residues and different lengths of runs of dG residues in the presence of sodium alone and in the presence of potassium and sodium. On the basis of the results, obtained primarily via solution-state NMR, it appears that very small loops favor parallel stranded quartet structures which do not require the presence of potassium. DNAs with loops of two to four residues and runs of two dG residues can form quadruplex structures of the "edge" or "chair" type in the presence of potassium but not in the presence of sodium alone. When all of the loops contain four residues, a "crossover" or "basket" type structure can be formed in the presence of sodium as well as in the presence of sodium and potassium. Structures with runs of three or four dG residues and with loops from two to four residues can form basket or crossover type structures in the absence of potassium. The presence of a purine in a loop can block both potassium binding and formation of chair type structures. Modeling of the interactions of cations with these quadruplex structures indicates that the potassium ions required for chair type structures interact with a terminal quartet and residues in the adjacent loop.     

Highly prevalent putative quadruplex sequence motifs in human DNA

We report here the results of a systematic search for the existence and prevalence of potential intramolecular G-quadruplex forming sequences in the human genome. We have also examined the tendency for particular sequences of ‘loop’ regions to occur in particular positions with respect to the G-tracts in a quadruplex. Using arithmetic ratio and probability techniques we have discovered frequent and systematic occurrence of certain sequence types, the most prominent being a potential quadruplex containing CCTGT in the first ‘loop’ position. Being able to highlight types of potential quadruplex sequences in G-rich regions is an important step in searching for biologically relevant sequences and finding their function.     

Sequence effects in single-base loops for quadruplexes

Intramolecular G-quadruplexes formed by a single DNA strand have attracted much interest due to the possibility that they may form in telomeres, oncogene promoter sequences and other biologically relevant regions of the genome. Therefore, it is important to understand the rules that govern the formation of these intramolecular structures and to determine their stabilities. We compared here 27 different sequences containing four tracts of three guanines with a medium (3) or relatively long (6-9 bases) central loop and two loops composed of a single nucleotide H (A, T or C) corresponding to the GGGHGGGN3-9GGGHGGG motif. These sequences are similar to sequence motifs that can be found in repeated and promoter sequences. Several conclusions were reached: (i) all sequences are prone to form very stable quadruplexes in potassium (Tm between 55 degrees C and 83 degrees C); (ii) these quadruplexes also form in sodium but with a strongly decreased Tm, with a 21 to 36 degrees C difference in melting temperature between Na+ and K+; (iii) a long (9 bases) central loop had a minimal deleterious impact on the stability of the quadruplex; (iv) pyrimidines are preferred over adenine in both single-base loops; (v) the folding topology is relatively robust in potassium: the CD spectra of all oligonucleotides matched the one of all-parallel stranded reference quadruplexes; (vi) conversely, in sodium the folding is diverse and sequence-dependent, as judged from CD and electrophoresis results.     

Effect of loop length variation on quadruplex-Watson Crick duplex competition

The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (Delta DeltaG(o)) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex.     

GG sequence of DNA and the human telomeric sequence react with cis-diammine-diaquaplatinum at comparable rates

G-quadruplex structures of telomeric sequences are of growing interest because they inhibit telomerase, an enzyme involved in the maintenance of telomere length of cancer cells. As we have shown previously, the antiparallel structure of G-quadruplexes can be cross-linked in vitro by the anti-tumour drug cisplatin. The question arises whether platination of quadruplex structures of human telomeric sequences by cisplatin could be relevant from a biological point of view. Therefore, we have compared the kinetics of reactions of the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), with the human telomeric quadruplex structure, a duplex DNA and a single-stranded DNA containing one specific platination GG site. The ratio between the platination rate constants was obtained using two intramolecular competition experiments: either a construct with a junction between duplex DNA containing a unique GG platination site and the quadruplex structure of the human telomeric sequence AG(3)(T(2)AG(3))(3), or a construct with a junction between duplex DNA and a single strand containing each a unique GG platination site. Those competition experiments allowed us to conclude that the platination of the quadruplex is favoured over that of the GG duplex by a factor of about two whereas the GG duplex is platinated three times faster than the GG single strand.     

Synthesis and characterization of monomolecular DNA G-quadruplexes formed by tetra-end-linked oligonucleotides

Guanine-rich DNA sequences are widely dispersed in the eukaryotic genome and are abundant in regions with relevant biological significance. They can form quadruplex structures stabilized by guanine quartets. These structures differ for number and strand polarity, loop composition, and conformation. We report here the syntheses and the structural studies of a set of interconnected d(TG(4)T) fragments which are tethered, with different orientations, to a tetra-end-linker in an attempt to force the formation of specific four-stranded DNA quadruplex structures. Two synthetic strategies have been used to obtain oligodeoxyribonucleotide (ODN) strands linked with their 3'- or 5'-ends to each of the four arms of the linker. The first approach allowed the synthesis of tetra-end-linked ODN (TEL-ODN) containing the four ODN strands with a parallel orientation, while the latter synthetic pathway led to the synthesis of TEL-ODNs each containing antiparallel ODN pairs. The influence of the linker at 3'- or 5'-ODN, on the quadruplex typology and stability, in the presence of sodium or potassium ions, has been investigated by circular dichroism (CD), CD thermal denaturation, (1)H NMR experiments at variable temperature, and molecular modeling. All synthesized TEL-ODNs formed parallel G-quadruplex structures. Particularly, the TEL-ODN containing all parallel ODN tracts formed very stable parallel G-quadruplex complexes, whereas the TEL-ODNs containing antiparallel ODN pairs led to relatively less stable parallel G-quadruplexes. The molecular modeling data suggested that the above antiparallel TEL-ODNs can adopt parallel G-quadruplex structures thanks to a considerable folding of the tetra-end-linker around the whole quadruplex scaffold.     

Structure of the Hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K⁺ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K⁺ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.     

Prevalence of quadruplexes in the human genome

Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, we present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. We count the number of quadruplexes found in the human genome and compare that with the figure predicted by modelling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. We demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the number of potentially relevant quadruplex-forming sequences. In particular, we show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.     

Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution

Intramolecular G-quadruplexes formed by human telomere sequences are attractive anticancer targets. Recently, four-repeat human telomere sequences have been shown to form two different intramolecular (3 + 1) G-quadruplexes in K(+) solution (Form 1 and Form 2). Here we report on the solution structures of both Form 1 and Form 2 adopted by natural human telomere sequences. Both structures contain the (3 + 1) G-tetrad core with one double-chain-reversal and two edgewise loops, but differ in the successive order of loop arrangements within the G-quadruplex scaffold. Our results provide the structural details at the two ends of the G-tetrad core in the context of natural sequences and information on different loop conformations. This structural information might be important for our understanding of telomere G-quadruplex structures and for anticancer drug design targeted to such scaffolds.     

Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC₅₀ values of 18–100 nM in a standard TRAP assay.     

Flexibility and structural conservation in a c-KIT G-quadruplex

A quadruplex sequence from the promoter region of the c-KIT gene forms a stable quadruplex, as characterized by crystallographic and NMR methods. Two new crystal structures are reported here, together with molecular dynamics simulation studies on these quadruplex crystal structures and an NMR structure. The new crystal structures, each in a distinct space group and lattice packing arrangement, together with the existing structures, demonstrate that the c-KIT quadruplex fold does not change with differing environments, suggesting that quadruplex topological dynamism is not a general phenomenon. The single and dinucleotide loops in these structures show a high degree of conformational flexibility within the three crystal forms and the NMR ensemble, with no evidence of clustering to particular conformers. This is in accord with the findings of high loop flexibility from the molecular dynamics studies. It is suggested that intramolecular quadruplexes can be grouped into two broad classes (i) those with at least one single-nucleotide loop, often showing singular topologies even though loops are highly flexible, and (ii) with all loops comprising at least two nucleotides, leading to topological dynamism. The loops can have more stable and less dynamic base-stacked secondary structures.     

Using principal component analysis to find correlations between loop-related and thermodynamic variables for G-quadruplex-forming sequences

The application of Principal Component Analysis (PCA) is proposed here as a simple means of revealing correlations between thermodynamic variables corresponding to folding equilibria of intramolecular G-quadruplexes and Watson–Crick duplexes, and the length of loops in the corresponding guanine-rich DNA sequences. To this end, two previously studied data sets were analyzed (Arora and Maiti, J. Phys. Chem. B. 2009 and Kumar and Maiti, Nucleic Acids. Res. 2008). All of the sequences considered shared the common structure 5’- GGG - loop1 - GGG - loop2 - GGG - loop3 - GGG -3’. PCA of these data sets supported a series of correlations between the variables studied. First, the association of loop length with thermodynamic stability and quadruplex structure was corroborated. Secondly, it is proposed that the addition of ethylene glycol produces a stronger stabilization on those sequences showing long loop1 and/or loop3. Thirdly, it is proposed that a low content of adenine in loop1 and/or loop3 will produce an increase in the stability of G-quadruplex and its related Watson–Crick duplex.     

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.