G-quadruplex motifs arranged in tandem occurring in telomeric repeats and the insulin-linked polymorphic region

To date, various G-quadruplex structures have been reported in the human genome. There are numerous studies focusing on quadruplex-forming sequences in general, but few studies have focused on two or more quadruplexes in the same molecule, which are most commonly found in telomeric DNA and other tandem repeats, e.g., insulin-linked polymorphic region (ILPR). Although the human telomere consists of a number of repeats, higher-order G-quadruplex structures are discussed less often because of the complexity of the structures. In this study, sequences consisting of 4-12 repeats of d(G(4)TGT), d(G(3)T(2)A), and/or d(G(4)T(2)A) have been studied by circular dichroism, ultraviolet spectroscopy, and temperature-gradient gel electrophoresis. These sequences serve as a model for the arrangement of quadruplexes in the telomere and ILPR in solution. Our major findings are as follows. (i) The number of G-rich repeats has a great influence on G-quadruplex stability. (ii) The evidence of quadruplex-quadruplex interaction is confirmed. (iii) For the first time, we directly observed the melting behavior of different conformers in a single experiment. Our results agree with other calorimetric and spectroscopic data and data obtained by single-molecule studies, atomic force microscopy, and mechanical unfolding by optical tweezers. We propose that the end of telomeres can be formed by only a few tandem quadruplexes (fewer than three). Our findings improve our understanding of the mechanism of G-quadruplex formation in long repeats in G-rich-regulating parts of genes and telomere ends.     

AC-motif: a DNA motif containing adenine and cytosine repeat plays a role in gene regulation

I-motif or C4 is a four-stranded DNA structure with a protonated cytosine:cytosine base pair (C+:C) found in cytosine-rich sequences. We have found that oligodeoxynucleotides containing adenine and cytosine repeats form a stable secondary structure at a physiological pH with magnesium ion, which is similar to i-motif structure, and have named this structure ‘adenine:cytosine-motif (AC-motif)’. AC-motif contains C⁺:C base pairs intercalated with putative A⁺:C base pairs between protonated adenine and cytosine. By investigation of the AC-motif present in the CDKL3 promoter (AC-motif_CDKL3), one of AC-motifs found in the genome, we confirmed that AC-motif_CDKL3 has a key role in regulating CDKL3 gene expression in response to magnesium. This is further supported by confirming that genome-edited mutant cell lines, lacking the AC-motif formation, lost this regulation effect. Our results verify that adenine-cytosine repeats commonly present in the genome can form a stable non-canonical secondary structure with a non-Watson–Crick base pair and have regulatory roles in cells, which expand non-canonical DNA repertoires.     

i-Motif DNA: Structure,stability and targeting with ligands

i-Motifs are four-stranded DNA secondary structures which can form in sequences rich in cytosine. Stabilised by acidic conditions, they are comprised of two parallel-stranded DNA duplexes held together in an antiparallel orientation by intercalated, cytosine–cytosine⁺ base pairs. By virtue of their pH dependent folding, i-motif forming DNA sequences have been used extensively as pH switches for applications in nanotechnology. Initially, i-motifs were thought to be unstable at physiological pH, which precluded substantial biological investigation. However, recent advances have shown that this is not always the case and that i-motif stability is highly dependent on factors such as sequence and environmental conditions. In this review, we discuss some of the different i-motif structures investigated to date and the factors which affect their topology, stability and dynamics. Ligands which can interact with these structures are necessary to aid investigations into the potential biological functions of i-motif DNA and herein we review the existing i-motif ligands and give our perspective on the associated challenges with targeting this structure.     

G-quadruplex and i-motif are mutually exclusive in ILPR double-stranded DNA

G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin.     

Evidence for intramolecularly folded i-DNA structures in biologically relevant CCC-repeat sequences.

The structural behaviour of repetitive cytosine DNA is examined in the oligodeoxynucleotide sequences of (CCCTAA)3CCCT (HTC4), GC(TCCC)3TCCT(TCCC)3 (KRC6) and the methylated (CCCT)3TCCT(CCCT)3C (KRM6) by circular dichroism (CD), gel electrophoresis (PAGE), and ultra violet (UV) absorbance studies. All the three sequences exhibit a pH-induced cooperative structural transition as monitored by CD. An intense positive CD band around 285 nm develops on lowering the pH from 8 to slightly acidic condition, indicative of the formation of base pairs between protonated cytosines. The oligomers are found to melt in a fully reversible and cooperative fashion, with a melting temperature (Tm) of around 50 degrees C at pH 5.5. The melting temperatures are independent from DNA concentration, indicative of an intramolecular process involved in the structural formation. PAGE experiments performed with 32P-labeled samples as well as with normal staining procedures show a predominantly single band migration for all the three oligomers suggestive of a unimolecular structure. From pH titrations the number of protons required for generating the structures formed by HTC4, KRC6 and KRM6 results to be around six. These findings strongly suggest that all the three sequences adopt an intramolecular i-motif structure. The demonstration of i-motif structure for KRC6, a critical functional stretch of the c-ki-ras promoter proto-oncogene, besides the human telomeric sequence HTC4, may be suggestive of larger significance in the functioning of DNA.     

Structure of a C-rich strand fragment of the human centromeric satellite III: a pH-dependent intercalation topology

Repetitive DNA sequences may adopt unusual pairing arrangements. At acid to neutral pH, cytidine-rich DNA oligodeoxynucleotides can form the i-motif structure in which two parallel-stranded duplexes with C.C(+) pairs are intercalated head-to-tail. The i-motif may be formed by multimeric associations or by intra-molecular folding, depending on the number of cytidine tracts, the nucleotide sequences between them, and the experimental conditions.We have found that a natural fragment of the human centromeric satellite III, d(CCATTCCATTCCTTTCC), can form two monomeric i-motif structures that differ in their intercalation topology and that are favored at pH values higher (the eta-form) and lower (the lambda-form) than 4.6. The change in intercalation may be related to adenine protonation in the loops.We studied the uridine derivative methylated on the first cytidine base, d(5mCCATTCCAUTCCUTTCC), whose proton spectrum is better resolved. The intercalation topologies are (C7.C17)/(5mC1.C11)/(C6.C16)/(C2.C12) for form lambda and (5mC1.C11)/(C7.C17)/(C2.C12)/(C6.C16) for form eta. We have solved the structure of the eta-form, and we present a model for the lambda-form. The switch from eta to lambda involves disruption of the i-motif. In both forms, the central AUT linker crosses the wide groove, and the first and the third linkers loop across the minor grooves. The i-motif core is extended in the eta-form by the inter-loop reverse Watson-Crick A3.U13 pair, whose dissociation constant is around 10(-2) at 0 degrees C, and in the lambda-form by the interloop T5.T15 pair.In contrast, d(5mCCATTCCTTACCTTTCC) folds into a pH-independent structure that has the same intercalation topology as the lambda-form. The i-motif core is extended below by the interloop T5.T15 pair and closed on top by the T8.A10 pair.Thus, the C-rich strand of the human satellite III tandem repeats, like the G-rich strand, can fold into various compact structures. The relevance of these features to centromeric function remains unknown.     

Human telomeric DNA: G-quadruplex,i-motif and Watson-Crick double helix

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson-Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C*C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding.     

A single trinucleotide, 5'AGC3'/5'GCT3', of the triplet-repeat disease genes confers metal ion-induced non-B DNA structure.

Expansion of (AGC)n repeats has been associated with genetic disorders called triplet-repeat diseases such as Huntington's disease (HD), myotonic muscular dystrophy (DM) and Kennedy's disease. To gain insight into the abnormal behavior of these repeats, we studied their structural properties in supercoiled DNA. Chemical probing revealed that, under physiological salt and pH conditions, Zn2+ or Co2+ ions induce (AGC)n repeats to adopt a novel non-B DNA structure in which all cytosine but none of adenine residues in either strand become unpaired. The minimum size of (AGC)n repeat that could form this structure independently of neighboring sequences is a single unit of double-stranded trinucleotide, 5'AGC3'/5'GCT3'. Other trinucleotide units of the same nucleotide composition, 5'CAG3'/5'CTG3' or 5'GCA3'/5'TGC3', do not form non-B DNA structures. This unusual DNA structural properly adopted by a single 5'AGC3'/5'GCT3' trinucleotide may contribute to expansion of (AGC)n sequences in triplet-repeat diseases.     

三核苷酸重复序列失稳定机制:重复序列形成non-B二级结构的必要性和细胞反式作用因子的作用

与三核苷酸重复序列CAG.CTG、CGG·CCG和GAA·TTC扩增和缺失有关的分子机制尚不能得到清楚的阐释.体外研究表明,上述疾病相关的重复序列可以在体外形成non-B二级结构,并介导重复序列扩增.然而,迄今为止,类似的观察尚未在体内研究过程中得以实现.利用模型生物大肠杆菌和酵母等进行的有关研究并不能模拟三核苷酸重复序列的扩增,这暗示三核苷酸重复序列的体内扩增可能与重复序列形成non-B二级结构关联性并不大.尽管理论上较长的三核苷酸重复序列可以在复制和后复制过程中较易形成non-B DNA二级结构,但这样的二级结构倾向于导致重复序列出现"脆性",而不是扩增.事实上,患者所具有的三核苷酸重复序列扩增并非一定需要通过non-B二级结构的介导,这些重复序列的扩增是可以通过一种RNA转录诱导的局部DNA重复序列的复制和其后的DNA重排得以发生.     

Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism

Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of (1)H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.     

Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA

Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3′ tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na⁺ and K⁺). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)₃ in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na⁺ to 3.40 at 15 mM Na⁺. Earlier spectral studies of Na⁺- and K⁺-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.     

Calorimetric unfolding of the bimolecular and i-motif complexes of the human telomere complementary strand, d(C(3)TA(2))(4)

A combination of spectroscopic and calorimetric techniques is used to determine the unfolding thermodynamics of the complexes formed by the complementary sequence of the human telomere, d(C(3)TA(2))(4), in the pH range of 4.2 to 6. Calorimetric melting curves show biphasic transitions; both transitions are shifted to higher temperatures as the pH is decreased, indicative of cytosine protonation, which favors the formation of C*C(+) base pairs. Furthermore, the transition temperature, T(M), of the lower transition depends on strand concentration, while the T(M) of the higher transition is independent of strand concentration, indicating the following sequential melting: bimolecular complex(s)-->intramolecular complex-->random coil. The thermodynamic profiles for the formation of each complex, bimolecular and i-motif reveals small favorable free energy terms resulting from favorable enthalpy-unfavorable entropy compensations, uptake of protons, marginal uptake of counterions (i-motif) and marginal release of water molecules (i-motif). Furthermore, an enthalpy of 3.2 kcal/mol (bimolecular complex) and 5.0 kcal/mol (i-motif) is estimated for a single C*C(+)/C*C(+) base-pair stack.     

The RNA i-motif

Oligodeoxynucleotides with stretches of cytidine residues associate into a four-stranded structure, the i-motif, in which two head-to-tail, intercalated, parallel-stranded duplexes are held together by hemiprotonated C.C+ pairs. We have investigated the possibility of forming an i-motif structure with C-rich ribonucleic acids. The four C-rich RNAs studied, r(UC5), r(C5), r(C5U) and r(UC3), associate into multiple intercalated structures at acidic pH. r(UC5) forms two i-motif structures that differ by their intercalation topologies. We report on a structural study of the main form and we analyze the small conformational differences found by comparison with the DNA i-motif. The stacking topology of the main structure avoids one of the six 2'-OH/2'-OH repulsive contacts expected in a fully intercalated structure. The C3'-endo pucker of the RNA sugars and the orientation of the intercalated C.C+ pairs result in a modest widening of the narrow grooves at the steps where the hydroxyl groups are in close contact. The free energy of the RNA i-motif, on average -4 kJ mol(-1) per C.C+ pair, is half of the value found in DNA i-motif structures.     

Recognition and elongation of telomeres by telomerase

Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase.     

Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.