Substitution of adenine for guanine in the quadruplex-forming human telomere DNA sequence G(3)(T(2)AG(3))(3)

We have studied the formation and structural properties of quadruplexes of the human telomeric DNA sequence G(3)(T(2)AG(3))(3) and related sequences in which each guanine base was replaced by an adenine base. None of these single base substitutions hindered the formation of antiparallel quadruplexes, as shown by circular dichroism, gel electrophoresis, and UV thermal stability measurements in NaCl solutions. Effect of substitution did differ, however, depending on the position of the substituted base. The A-for-G substitution in the middle quartet of the antiparallel basket scaffold led to the most distorted and least stable structures and these sequences preferred to form bimolecular quadruplexes. Unlike G(3)(T(2)AG(3))(3), no structural transitions were observed for the A-containing analogs of G(3)(T(2)AG(3))(3) when sodium ions were replaced by potassium ions. The basic quadruplex topology remained the same for all sequences studied in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may have biological relevance.     

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G₃(TTAG₃)₃ forms an antiparallel quadruplex of the same basket type in solution containing either K⁺ or Na⁺ ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K⁺-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G₃(TTAG₃)₃ motif. Both G₃(TTAG₃)₃ and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K⁺-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K⁺-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG₃(TTAG₃)₃ by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K⁺ ions.     

The abasic site lesions in the human telomeric sequence d[TA(G3T2A)3G3]: A thermodynamic point of view

Background

The abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes.

Methods

In this paper, we report investigations, based on calorimetric, UV, CD and electrophoretic techniques, on 12 oligodeoxynucleotides analogues of the quadruplex forming human telomere sequence d[TA(G₃T₂A)₃G₃], in which each guanine has been replaced, one at a time, by an abasic site mimic.

Results

Although all data show that the modified sequences preserve their ability to form quadruplex structures, the thermodynamic parameters clearly indicate that the presence of an abasic site decreases their thermal stability compared to the parent unmodified sequence, particularly if the replacement concerns one of the guanosines involved in the formation of the central G-tetrad.

Conclusions

The collected data indicate that the effects of the presence of abasic site lesions in telomeric quadruplex structures are site-specific. The most dramatic consequences come out when this lesion involves a guanosine in the centre of a G-run.

General significance

Abasic sites, by facilitating the G-quadruplex disruption, could favour the formation of the telomerase primer. Furthermore they could have implications in the pharmacological approach targeting telomere.     

Folding thermodynamics of the hybrid‐1 type intramolecular human telomeric G‐quadruplex

Guanine‐rich DNA sequences that may form G‐quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G‐quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G‐quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G‐rich sequences in the presence or the absence of their complementary C‐rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26‐meric human telomeric sequence d[A₃G₃(T₂AG₃)₃A₂]. In the presence of K⁺ ions, the latter adopts the hybrid‐1 G‐quadruplex conformation, a tightly packed structure with an unusually small number of solvent‐exposed atomic groups. The K⁺‐induced folding of the G‐quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G‐quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G‐quadruplex formation. Based on our volume data, 432 ± 19 water molecules become released to the bulk upon the G‐quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute–solvent interactions all over the surface of the folded structure. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014.     

Covalent ligation studies on the human telomere quadruplex

Recent X-ray crystallographic studies on the human telomere sequence d[AGGG(TTAGGG)₃] revealed a unimolecular, parallel quadruplex structure in the presence of potassium ions, while earlier NMR results in the presence of sodium ions indicated a unimolecular, antiparallel quadruplex. In an effort to identify and isolate the parallel form in solution, we have successfully ligated into circular products the single-stranded human telomere and several modified human telomere sequences in potassium-containing solutions. Using these sequences with one or two terminal phosphates, we have made chemically ligated products via creation of an additional loop. Circular products have been identified by polyacrylamide gel electrophoresis, enzymatic digestion with exonuclease VII and electrospray mass spectrometry in negative ion mode. Optimum pH for the ligation reaction of the human telomere sequence ranges from 4.5 to 6.0. Several buffers were also examined, with MES yielding the greatest ligation efficiency. Human telomere sequences with two phosphate groups, one each at the 3′ and 5′ ends, were more efficient at ligation, via pyrophosphate bond formation, than the corresponding sequences with only one phosphate group, at the 5′ end. Circular dichroism spectra showed that the ligation product was derived from an antiparallel, single-stranded guanine quadruplex rather than a parallel single-stranded guanine quadruplex structure.     

The four repeat Giardia lamblia telomere forms tetramolecular G-quadruplex with antiparallel topology

Abstract

Guanine rich DNA sequences of regulatory genomic regions form secondary structures known as G-quadruplexes usually stabilized by tetrads of Hoogsteen hydrogen bonded guanines. The in vivo existence of G-quadruplexes ascertains their biological roles. Human telomeric repeats are the most studied G-rich sequences. The four repeat Giardia telomeric sequence (TAGGG)₄ differs from its human counterpart (TTAGGG)_4, by deletion of one T at the G-tract intervening site of each repeat. We show here that whilst the two repeat Giardia telomeric sequence (TAGGG)₂ forms parallel and antiparallel quadruplexes with tetramolecular topology exclusively, the four repeat version (TAGGG)₄ forms a tetramolecular (antiparallel) and unimolecular (parallel) quadruplexes in Na⁺. The tetramolecular (antiparallel) G-quadruplex formed by four repeats of Giardia telomeric sequence is stabilized by the additional Watson-Crick bonding between its intervening TA bases aligned in antiparallel fashion. Four stranded antiparallel quadruplex for four repeats of any telomeric sequence have not been characterized till date. We hypothesize that telomeric association in antiparallel fashion, (via G-overhangs to form tetramolecular quadruplex) could be a biologically relevant molecular event. Further, coexistence of Hoogsteen as well as Watson-Crick base pairing might give insight for recognition of conformationally diverse DNA structures by ligands.

Communicated by Ramaswamy H. Sarma     

Natural isoflavones regulate the quadruplex–duplex competition in human telomeric DNA

Effects of natural isoflavones on the structural competition of human telomeric G-quadruplex d[AG₃(T₂AG₃)₃] and its related Watson–Crick duplex d[AG₃(T₂AG₃)₃-(C₃TA₂)₃C₃T] are investigated by using circular dichroism (CD), ESI-MS, fluorescence quenching measurement, CD stopped-flow kinetic experiment, UV spectroscopy and molecular modeling methods. It is intriguing to find out that isoflavones can stabilize the G-quadruplex structure but destabilize its corresponding Watson–Crick duplex and this discriminated interaction is intensified by molecular crowding environments. Kinetic experiments indicate that the dissociation rate of quadruplex (k_{obs290 nm}) is decreased by 40.3% at the daidzin/DNA molar ratio of 1.0 in K⁺, whereas in Na⁺ the observed rate constant is reduced by about 12.0%. Furthermore, glycosidic daidzin significantly induces a structural transition of the polymorphic G-quadruplex into the antiparallel conformation in K⁺. This is the first report on the recognition of isoflavones with conformational polymorphism of G-quadruplex, which suggests that natural isoflavone constituents potentially exhibit distinct regulation on the structural competition of quadruplex versus duplex in human telomeric DNA.     

Secondary structure polymorphism in Oxytricha nova telomeric DNA

Tandem repeats of the telomeric DNA sequence d(T₄G₄) of Oxytricha nova are capable of forming unusually stable secondary structures incorporating Hoogsteen hydrogen bonding interactions. The biological significance of such DNA structures is supported by evidence of specific recognition of telomere end-binding proteins in the crystal state. To further characterize structural polymorphism of Oxytricha telomeric DNAs, we have obtained and interpreted Raman, ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectra of the tandem repeats d(G₄T₄G₄) (Oxy1.5), d(T₄G₄)₂ (Oxy2) and dT₆(T₄G₄)₂ (T6Oxy2) and related non-telomeric isomers in aqueous salt solutions. Raman markers of Oxy1.5 identify both C2′-endo/anti and C2′-endo/syn conformations of the deoxyguanosine residues and Hoogsteen hydrogen bonded guanine quartets, consistent with the quadruplex fold determined previously by solution NMR spectroscopy. Raman, UVRR and CD signatures and Raman dynamic measurements, to monitor imino NH→ND exchanges, show that the Oxy1.5 antiparallel quadruplex fold is distinct from the hairpin structures of Oxy2 and T6Oxy2, single-stranded structures of d(TG)₈ and dT₆(TG)₈ and previously reported quadruplex structures of d(T₄G₄)₄ (Oxy4) and dG₁₂. Spectral markers of the telomeric and telomere-related DNA structures are tabulated and novel Raman and UVRR indicators of thymidine and deoxyguanosine conformations are identified. The results will be useful for probing structures of Oxytricha telomeric repeats in complexes with telomere end-binding proteins.     

Interaction between cationic zinc porphyrin and lead ion induced telomeric guanine quadruplexes: evidence for end-stacking

The interaction between small molecules and telomeric quadruplex DNA has received great attention because of its importance in molecular recognition and anticancer drug design. Using UV/vis absorption titration, thermal melting, circular dichroism spectroscopy, and electrospray ionization mass spectrometry, we examined the formation of lead ion induced guanine quadruplexes (Pb-G4) from oligonucleotide AG₃(T₂AG₃)₃ and their interaction with a zinc derivative of 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (Zn-TMPyP). The binding of lead ion to the oligonucleotide was found to have an unusually high affinity and followed a 1:1 stoichiometry, and the resultant Pb-G4 structure was stabilized by Zn-TMPyP binding. Owing to the steric hindrance of the axial ligand of zinc and also the relatively rigid structure of Pb-G4, intercalation of Zn-TMPyP between adjacent guanine quartets is precluded, thus allowing the end-stacking binding mode to be characterized exclusively. In conjunction with a big redshift (more than 8 nm) in the absorption spectrum, we demonstrate that a conservative induced circular dichroism is an important signature for end-stacking of porphyrins on guanine quadruplexes.     

Effect of Cationic Comb-Type Copolymer on Quadruplex Folding of Human Telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA) _n in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA) ₃ GGG) and d((GGGTTA)₇GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA) ₃ GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions

The quadruplex structures of the human telomere sequences AG₃(T₂AG₃)₃ I and (T₂AG₃)₄ II were investigated in the presence of Na⁺ and K⁺ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH₃)₂ moieties were identified by chemical and 3′-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na⁺ solution and B from X-ray data of a K⁺-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1–G10, A13–G22, A1–A13 platinum chelates at the top of the quadruplex and A7–A19, G4–A19 and A7–G20 at the bottom, whether the Na⁺ or K⁺ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na⁺ and K⁺ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.     

Intramolecular quadruplex conformation of human telomeric DNA assessed with 125I-radioprobing

A repeated non-coding DNA sequence d(TTAGGG)_n is present in the telomeric ends of all human chromosomes. These repeats can adopt multiple inter and intramolecular non-B-DNA conformations that may play an important role in biological processes. Two intramolecular structures of the telomeric oligonucleotide dAGGG(TTAGGG)₃, antiparallel and parallel, have been solved by NMR and X-ray crystallography. In both structures, the telomeric sequence adopts an intramolecular quadruplex structure that is stabilized by G-4 quartets, but the ways in which the sequence folds into the quadruplex are different. The folds of the human telomeric DNA were described as an anti-parallel basket-type and a parallel propeller-type. We applied ¹²⁵I-radioprobing to determine the conformation of the telomeric quadruplex in solution, in the presence of either Na⁺ or K⁺ ions. The probability of DNA breaks caused by decay of ¹²⁵I is inversely related to the distance between the radionuclide and the sugar unit of the DNA backbone; hence, the conformation of the DNA backbone can be deduced from the distribution of breaks. The probability of breaks measured in the presence of Na⁺ and K⁺ were compared with the distances in basket-type and propeller-type quadruplexes obtained from the NMR and crystal structures. Our radioprobing data demonstrate that the antiparallel conformation was present in solution in the presence of both K⁺ and Na⁺. The preferable conformation in the Na⁺-containing solution was the basket-type antiparallel quadruplex whereas the presence of K⁺ favored the chair-type antiparallel quadruplex. Thus, we believe that the two antiparallel and the parallel conformations may coexist in solution, and that their relative proportion is determined by the type and concentration of ions.     

Intramolecular DNA quadruplexes with different arrangements of short and long loops

We have examined the folding, stability and kinetics of intramolecular quadruplexes formed by DNA sequences containing four G₃ tracts separated by either single T or T₄ loops. All these sequences fold to form intramolecular quadruplexes and 1D-NMR spectra suggest that they each adopt unique structures (with the exception of the sequence with all three loops containing T₄, which is polymorphic). The stability increases with the number of single T loops, though the arrangement of different length loops has little effect. In the presence of potassium ions, the oligonucleotides that contain at least one single T loop exhibit similar CD spectra, which are indicative of a parallel topology. In contrast, when all three loops are substituted with T₄ the CD spectrum is typical of an antiparallel arrangement. In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure. Kinetic studies on the complexes with one or two T₄ loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.     

Studies on Non-Covalent Interaction of Coumarin Attached Pyrimidine and 1-Methyl Indole 1,2,3 Triazole Analogues with Intermolecular Telomeric G-Quadruplex DNA Using ESI-MS and Spectroscopy

In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T₂AG₃) and d(T₂AG₃)₂ sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV–Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T₂AG₃) and d(T₂AG₃)₂ G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.     

Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A,G:G:A:A,and A:A:A:A tetrads

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG₃(TTAG₃)₃ did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three‐tetrad scaffold, as proved by CD and PAGE in both Na⁺ and K⁺ solutions. Thermodynamic data showed that, in Na⁺ solution, the dG₃(TTAG₃)₃ quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced syn‐guanosines. In physiological K⁺ solution, the highest destabilization was caused by the 4A tetrad. In K⁺, only the unmodified dG₃(TTAG₃)₃ quadruplex rearranged into a K⁺‐dependent quadruplex form, none of the multiple adenine‐modified structures did so. This may imply biological consequences for nonrepaired A‐for‐G mutations. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 880–886, 2010.     

Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T₂AG₃)₄ (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T₂AG₃)₁₃ (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)₇ and d(GAAA)₅, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.     

8-oxoguanine in a quadruplex of the human telomere DNA sequence

8-Oxoguanine is a ubiquitous oxidative base lesion. We report here on the effect of this lesion on the structure and stability of quadruplexes formed by the human telomeric DNA sequence 5'-dG(3)(TTAG(3))(3) in NaCl and KCl. CD, PAGE and absorption-based thermodynamic stability data showed that replacement of any of the tetrad-forming guanines by 8-oxoguanine did not hinder the formation of monomolecular, antiparallel quadruplexes in NaCl. The modified quadruplexes were, however, destabilized in both salts, the extent of this depending on the position of the lesion. These results and the results of previous studies on guanine-to-adenine exchanges and guanine abasic lesions in the same quadruplex show a noticeable trend: it is not the type of the lesion but the position of the modification that determines the effect on the conformation and stability of the quadruplex. The type of lesion only governs the extent of changes, such as of destabilization. Most sensitive sites were found in the middle tetrad of the three-tetrad quadruplex, and the smallest alterations were observed if guanines of the terminal tetrad with the diagonal TTA loop were substituted, although even these substitutions brought about unfavorable enthalpic changes. Interestingly, the majority of these base-modified quadruplexes did not adopt the rearranged folding induced in the unmodified dG(3)(TTAG(3))(3) by potassium ions, an observation that could imply biological relevance of the results.     

Stoichiometry-driven Protein Folding: A Comment

Abstract

We have found, with the aid of 2-D gel electrophoresis, that double-stranded human telomeric repeat, (T₂AG₃)₁₂·(C₃TA₂)₁₂, being cloned within a plasmid, forms a protonated superhelically-induced structure. Experiments on chemical and enzymatic probing also indicate that the human telomeric repeats adopt an unusual structure. We have proposed an eclectic model for this structure in which four different elements coexist: a non-orthodox intramolecular triplex stabilized by the canonical protonated C · G*C⁺ base-triads and highly enriched by non-canonical base-triads; the intramolecular quadruplex formed by a portion of the G-rich strand; the single-stranded region encompassing a portion of the G-rich strand and, probably, the (C,A)-hairpin formed by a portion of the C-rich strand.     

Unusual behavior exhibited by multistranded guanine-rich DNA complexes

The structural properties of oligonucleotides containing two different types of G-rich sequences at the 3′-ends were compared. It is shown that oligonucleotides with uninterrupted runs of guanine residues at the 3′-end, e.g., d(T₁₅G₁₂), form multistranded structures stabilized by guanine-guanine interactions. The chemical and physical properties of these complexes differ from those of the complexes formed by oligonucleotides with telomere-like sequences, e.g., d(T₁₅G₄T₂G₄). In methylation protection and methylation interference experiments, we found all the guanines in complexes formed by d(T₁₅G₁₅) and d(T₁₅G₁₂) to be accessible to methylation. Furthermore, the methylated monomers retain the ability to polymerize. This contrasts with the inaccessibility of the guanines in d(T₁₅G₄T₂G₄) to methylation and the inability of the methylated monomer to form supramolecular structures. The stoichiometry of the complexes arising from the two types of oligonucleotides also differs. The complexes formed by d(T₁₅G₁₅) consist of consecutive integer numbers of DNA strands, whereas complexes formed by telomere-like oligonucleotides contain 1, 2, 4, or multiples of four strands. Magnesium ions favor formation of high molecular weight complexes by d(T₁₅G₁₅) and d(T₁₅G₁₂), but not by d(T₁₅G₄T₂G₄). The d(T₁₅G₁₅) and d(T₁₅G₁₂) complexes have very high thermal stability compared with telomeric complexes. However, at low temperatures, the thymine bases within the telomeric motif, TTGGGGTTGGGG, appear to allow for the formation of stable high-molecular weight species with a longer nonguanine portion. © 1998 John Wiley & Sons, Inc. Biopoly 45: 427–434, 1998