Antiparallel Triplex Research Articles

Detection of pyrimidine-rich DNA sequences based on the formation of parallel and antiparallel triplex DNA and fluorescent silver nanoclusters

In this work, the use of DNA-stabilized fluorescent silver nanoclusters for the detection of target pyrimidine-rich DNA sequences by formation of parallel and antiparallel triplex structures is studied by molecular fluorescence spectroscopy. In the case of parallel triplexes, the probe DNA fragments are Watson-Crick stabilized hairpins, and whereas in the case of antiparallel triplexes, the probe fragments are reverse-Hoogsteen clamps. In all cases, the formation of the triplex structures has been assessed by means of polyacrylamide gel electrophoresis, circular dichroism, and molecular fluorescence spectroscopies, as well as multivariate data analysis methods. The results have shown that it is possible the detection of pyrimidine-rich sequences with an acceptable selectivity by using the approach based on the formation of antiparallel triplex structures.

Recurrent Potential G-Quadruplex Sequences in Archaeal Genomes.

Evolutionary conservation or over-representation of the potential G-quadruplex sequences (PQS) in genomes are usually considered as a sign of the functional relevance of these sequences. However, uneven base distribution (GC-content) along the genome may along the genome may result in seeming abundance of PQSs over average in the genome. Apart from this, a number of other conserved functional signals that are encoded in the GC-rich genomic regions may inadvertently result in emergence of G-quadruplex compatible sequences. Here, we analyze the genomes of archaea focusing our search to repetitive PQS (rPQS) motifs within each organism. The probability of occurrence of several identical PQSs within a relatively short archaeal genome is low and, thus, the structure and genomic location of such rPQSs may become a direct indication of their functionality. We have found that the majority of the genomes of Methanomicrobiaceae family of archaea contained multiple copies of the interspersed highly similar PQSs. Short oligonucleotides corresponding to the rPQS formed the G-quadruplex (G4) structure in presence of potassium ions as demonstrated by circular dichroism (CD) and enzymatic probing. However, further analysis of the genomic context for the rPQS revealed a 10–12 nt cytosine-rich track adjacent to 3'-end of each rPQS. Synthetic DNA fragments that included the C-rich track tended to fold into alternative structures such as hairpin structure and antiparallel triplex that were in equilibrium with G4 structure depending on the presence of potassium ions in solution. Structural properties of the found repetitive sequences, their location in the genomes of archaea, and possible functions are discussed.

Detection of parallel and antiparallel DNA triplex structures in living human cells using in-cell NMR.

We introduced oligodeoxynucleotides (ODNs) that form parallel and antiparallel triplex structures in vitro into living human cells and recorded their in-cell NMR spectra. Observation of landmark signals for triplex structures proved for the first time that parallel and antiparallel triplex structures are formed in living human cells.

The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics.

A telomere carrying repetitive sequences ends with a single-stranded overhang. The G-rich overhang could fold back and bind in the major groove of its upstream duplex, forming an antiparallel triplex structure. The telomeric triplex has been proposed to function in protecting chromosome ends. However, we lack strategies to mechanically probe the dynamics of a telomeric triplex. Here, we show that the topological dynamics of a telomeric triplex involves 3′ overhang binding at the ds/ssDNA junction inferred by DNA mechanics. Assisted by click chemistry and branched polymerase chain reaction, we developed a rescue-rope-strategy for mechanically manipulating an artificial telomeric DNA with a free end. Using single-molecule magnetic tweezers, we identified a rarely forming (5%) telomeric triplex which pauses at an intermediate state upon unzipping the Watson–Crick paired duplex. Our findings revealed that a mechanically stable triplex formed in a telomeric DNA can resist a force of 20 pN for a few seconds in a physiological buffer. We also demonstrated that the rescue-rope-strategy assisted mechanical manipulation can directly rupture the interactions between the third strand and its targeting duplex in a DNA triplex. Our single-molecule rescue-rope-strategy will serve as a general tool to investigate telomere dynamics and further develop triplex-based biotechnologies.

Monitoring the Structure-Dependent Reaction Pathways of Guanine Radical Cations in Triplex DNA: Deprotonation Versus Hydration.

Exposure of DNA to one-electron oxidants leads initially to the formation of guanine radical cations (G•+), which may degrade by deprotonation or hydration and ultimately cause strand breaks or 8-oxoG lesions. As the structure is dramatically changed by binding of the third strand in the major groove of the target duplex, it makes the triplex an interesting DNA structure to be examined and compared with the duplex on the G•+ degradation pathways. Here, we report for the first time the time-resolved spectroscopy study on the G•+ reaction dynamics in triplex DNA together with the Fourier transform infrared characterization of steady-state products, from which structural effects on the reactivity of G•+ are unraveled. For an antiparallel triplex-containing GGC motif, G•+ mainly suffers from fast deprotonation (9.8 ± 0.2) × 106 s-1, featuring release of both N1-H and N2-H of G in the third strand directly into bulk water. The much faster and distinct deprotonation behavior compared to the duplex should be related to long-resident water spines in the third strand. The G•+ hydration product 8-oxoG is negligible for an antiparallel triplex; instead, the 5-HOO-(G-H) hydroperoxide formed after G•+ deprotonation is identified by its vibrational marker band. In contrast, in a parallel triplex (C+GC), the deprotonation of G•+ occurs slowly (6.0 ± 0.3) × 105 s-1 with the release of N1-H, while G•+ hydration becomes the major pathway with yields of 8-oxoG larger than in the duplex. The increased positive charge brought by the third strand makes the G radical in the parallel triplex sustain more cation character and prone for hydration. These results indicate that non-B DNA (triplex) plays an important role in DNA damage formation and provide mechanistic insights to rationalize why triplex structures might become hot spots for mutagenesis.

Stable and Selective Antiparallel Type Triplex DNA Formation by Targeting a GC Base Pair with the TFO Containing One <i>N</i><sup>2</sup>-Phenyl-2′-deoxyguanosine

The antiparallel triplex DNA is formed by the interaction between purine-rich triplex forming oligonucleotides (TFOs) and the homo-purine region within a duplex DNA. The formation of such a structure with the genome DNA promises to control the gene expression in a living cell. In this study, in an attempt to enhance the stability of the triplex DNAs, we have designed the N2-arylated deoxyguanosine derivatives. Among these analogues, we found that the TFOs containing N2-phenyl-2'-deoxyguanosine (PhdG) showed a stable and selective triplex DNA formation with the GC base pair as compared to the natural dG/GC triplet. However, the multiple incorporation of PhdG into the TFOs hampered the stable triplex DNA, instead, showed a tendency to form a higher order structure. Therefore, we concluded that the stable and selective triplex DNA formation is expected by the replacement of dG by PhdG in the purine-rich TFO sequence.

Effect of the 3-halo substitution of the 2′-deoxy aminopyridinyl-pseudocytidine derivatives on the selectivity and stability of antiparallel triplex DNA with a CG inversion site

Triplex formation against a target duplex DNA has the potential to become a tool for the genome research. However, there is an intrinsic restriction on the duplex DNA sequences capable of forming the triplex DNA. Recently, we demonstrated the selective formation of the stable antiparallel triplexes containing the CG inversion sites using the 2′-deoxy-1-methylpseudocytidine derivative (ΨdC), whose amino group was conjugated with the 2-aminopyridine at its 5-position as an additional hydrogen bonding unit (AP-ΨdC). The 1-N of 2-aminopyridine was supposed to be protonated to form the hydrogen bond with the guanine of the CG inversion site. In this study, to test the effect of the 3-substitution of the 2-aminopyridine unit of AP-ΨdC on the triplex stability, we synthesized the 3-halogenated 2-aminopyridine derivatives of AP-ΨdC. The pKa values 1-N of the 2-aminopyridine unit of AP-ΨdC as the monomer nucleoside were determined to be 6.3 for 3-CH3 (MeAP-ΨdC), 6.1 for 3-H (AP-ΨdC), 4.3 for 3-Cl (ClAP-ΨdC), 4.4 for 3-Br (BrAP-ΨdC), and 4.7 for 3-I (IAP-ΨdC), suggesting that all the halogenated AP-ΨdCs are not protonated under neutral conditions. Interestingly, although the recognition selectivity depends on the sequence context, the TFO having the sequence of the 3′-G-(IAP-ΨdC)-A-5′ context showed the selective triplex formation with the CG inversion site. These results suggest that the protonation at the 1-N position plays an important role in the stable and selective triplex formation of AP-ΨdC derivatives in any sequences.

Aminopyridinyl-Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites.

The sequence-specific formation of triplex DNA offers a potential basis for genome-targeting technologies. In an antiparallel triplex DNA, the sequence-specificity is established by the formation of specific base triplets (G-GC, A-AT, and T-AT) between a triplex-forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long-standing goal for the triplex-forming technology. We now describe the design and synthesis of pseudo-deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex-forming sequence. The aminopyridine-bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3-methyl-2-aminopyridinyl-ΨdC ((Me) AP-ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, (Me) AP-ΨdC is expected to serve as a new starting point of triplex-forming oligonucleotides for a wide variety of genome-targeting applications.

Aminopyridinyl–Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites

AbstractThe sequence‐specific formation of triplex DNA offers a potential basis for genome‐targeting technologies. In an antiparallel triplex DNA, the sequence‐specificity is established by the formation of specific base triplets (G−GC, A−AT, and T−AT) between a triplex‐forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long‐standing goal for the triplex‐forming technology. We now describe the design and synthesis of pseudo‐deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex‐forming sequence. The aminopyridine‐bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3‐methyl‐2‐aminopyridinyl−ΨdC (MeAP−ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, MeAP−ΨdC is expected to serve as a new starting point of triplex‐forming oligonucleotides for a wide variety of genome‐targeting applications.

Anti-parallel triplexes: Synthesis of 8-aza-7-deazaadenine nucleosides with a 3-aminopropynyl side-chain and its corresponding LNA analog

The phosphoramidites of DNA monomers of 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine (Y) and 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine LNA (Z) are synthesized, and the thermal stability at pH 7.2 and 8.2 of anti-parallel triplexes modified with these two monomers is determined. When, the anti-parallel TFO strand was modified with Y with one or two insertions at the end of the TFO strand, the thermal stability was increased 1.2°C and 3°C at pH 7.2, respectively, whereas one insertion in the middle of the TFO strand decreased the thermal stability 1.4°C compared to the wild type oligonucleotide. In order to be sure that the 3-aminopropyn-1-yl chain was contributing to the stability of the triplex, the nucleobase X without the aminopropynyl group was inserted in the same positions. In all cases the thermal stability was lower than the corresponding oligonucleotides carrying the 3-aminopropyn-1-yl chain, especially at the end of the TFO strand. On the other hand, the thermal stability of the anti-parallel triplex was dramatically decreased when the TFO strand was modified with the LNA monomer analog Z in the middle of the TFO strand (ΔTm=−9.1°C). Also the thermal stability decreased about 6.1°C when the TFO strand was modified with Z and the Watson–Crick strand with adenine-LNA (AL). The molecular modeling results showed that, in case of nucleobases Y and Z a hydrogen bond (1.69 and 1.72Ǻ, respectively) was formed between the protonated 3-aminopropyn-1-yl chain and one of the phosphate groups in Watson–Crick strand. Also, it was shown that the nucleobase Y made a good stacking and binding with the other nucleobases in the TFO and Watson–Crick duplex, respectively. In contrast, the nucleobase Z with LNA moiety was forced to twist out of plane of Watson–Crick base pair which is weakening the stacking interactions with the TFO nucleobases and the binding with the duplex part.

An isocytidine derivative with a 2-amino-6-methylpyridine unit for selective recognition of the CG interrupting site in an antiparallel triplex DNA.

Sequence-specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non-natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N(2)-arylmethyl isodC derivatives and incorporated them into triplex-forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2-amino-6-methylpyridine moiety (AP-isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.

The DNA-forming properties of 6-selenoguanine

We present here an exhaustive characterization of the structure and properties of 6-selenoguanine, an isoster of guanine, and the impact of its introduction in DNA. This study reports the results of state-of-the-art quantum mechanical calculations and atomistic molecular dynamics simulations carried out to shed light on the impact of the replacement of guanine (G) by 6-selenoguanine (SeG) in different forms of DNA. The results point out that the G → SeG substitution leads to stable DNA duplex, antiparallel triplex and G-quadruplex structures, though local distortions are also found. These structural changes affect the thermodynamic stability of the mutation leading to a clear destabilization for all studied systems. Interestingly, the lowest effect has been found when the mutation was placed in the triplex-forming oligonucleotide strand in a reverse Hoogsteen orientation, which favours the antiparallel triplex formation regarding the G-tetraplex formation. Detailed QM studies strongly suggest that SeG impacts the HOMO-LUMO gap and accordingly the transfer properties of DNA, opening the way to modulate the conductivity properties of non-natural DNAs.

Spectroscopic and Calorimetric Studies on the Binding of an Indoloquinoline Drug to Parallel and Antiparallel DNA Triplexes

11-Phenyl-substituted indoloquinolines have been found to exhibit significant antiproliferative potency in cancer cells but to show only moderate affinity toward genomic double-helical DNA. In this study, parallel as well as antiparallel triple-helical DNA targets are employed to evaluate the triplex binding of these ligands. UV melting experiments with parallel triplexes indicate considerable interactions with the drug and a strong preference for TAT-rich triplexes in line with an increasing number of potential intercalation sites of similar binding strength between two TAT base triads. Via substitution of a singly charged aminoethylamine side chain by a longer and doubly charged bis(aminopropyl)amine substituent at the ligand, binding affinities increase and also start to exhibit long-range effects as indicated by a strong correlation between the binding affinity and the overall length of the TAT tract within the triplex stem. Compared to parallel triplexes, an antiparallel triplex with a GT-containing third strand constitutes a preferred target for the indoloquinoline drug. On the basis of pH-dependent titration experiments and corroborated by a Job analysis of continuous variation, binding of the drug to the GT triplex not only is strongly enhanced when the solution pH is lowered from 7 to 5 but also reveals a pH-dependent stoichiometry upon formation of the complex. Calorimetric data demonstrate that stronger binding of a protonated drug at acidic pH is associated with a more exothermic binding process. However, at pH 7 and 5, binding is enthalpically driven with additional favorable entropic contributions.

Synthesis of 1′-phenyl-2′-OMe ribose analogues connecting the thymine base at the 1′ position through a flexible linker for the formation of a stable anti-parallel triplex DNA

We have previously developed the innovative bicyclic nucleoside analogues (WNA) for the formation of the triplex DNA. The WNA analogue consists of an aromatic ring and a recognition base on the bicyclic skeleton, and the recognition of the CG or TA interrupting sites has been achieved by the WNA analogues. However, the stabilization ability of the WNA analogue is dependent on its neighboring nucleobases within the TFO. We hypothesized that the sequence dependency might arise from the fixed conformation of the bicyclic ring of the WNA. Thus, it was expected that an open-linker between the sugar part and the nucleobase might produce the flexibility and improve the stabilizing effect of the nucleobase analogues. We now report the design and synthesis of a new nucleoside analogue as an open-form of WNA-βT, the 1′-phenyl-2′-OMe-ribose derivative, connecting the thymine base to the ribose part through a methylene linker (1) or an ethylene linker (2). TFO containing the 3′-dA-1-dG context recognized the CG interrupting site, and that with the 3′-dG-1-dG context recognized the GC site. In contrast, 2 displayed a stabilizing effect on all four base pairs with some preferences for the TFO containing 3′-dA-2-dG and 3′-dG-2-dG. These results suggested that a flexible linker between the nucleobase and the ribose part may improve the sequence dependency for the triplex formation.

An efficient antigene activity and antiproliferative effect by targeting the Bcl-2 or survivin gene with triplex forming oligonucleotides containing a W-shaped nucleoside analogue (WNA-βT)

Triplex forming oligonucleotides (TFOs) are some of the most promising tools in the antigene strategy for the development of gene targeting therapeutics. However, the stable triplex formation is restricted to the homopurine sequences consisting of purine nucleosides, dG and dA. Therefore, the T or dC nucleoside in the homopurine strand inhibits the stable triplex formation. We have developed W-shaped nucleoside analogues (WNAs) for the formation of the unnatural type triplex DNA, with sequences containing the interrupting site in an antiparallel triplex formation. In the present study, we tested the antigene effect of TFOs having WNA-βT, which increased the stability of the triplex formation with a target sequence including the TA interrupting site. We designed the GU TFO (WNA) and GU TFO (natural) for targeting sequences of the Bcl-2 or survivin oncogene. The gel shift assay showed that the TFO (WNA) formed more stable triplexes than the natural TFO. Remarkably, the Bcl-2- or survivin-targeted TFO (WNA) inhibited the cell proliferation and induced a caspase-dependent apoptosis. It was confirmed that the survivin-targeted TFO (WNA) more effectively decreased the number of survivin products in the A549 cell than the natural TFOs.

Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA

We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid steric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was illustrated by the fact that the TFO containing WNA-βAP in the 3′G-WNA-G-5′ sequence formed a stable triplex with selectivity to the CG interrupting site where the previous WNA-βC did not induce the triplex formation.

Synthesis of p-amino-WNA derivatives to enhance the stability of the anti-parallel triplex

We have previously developed W-shaped nucleoside analogs (WNAs) having a nucleobase and an aromatic ring for the formation of the unnatural triplex DNA. Modification of an aromatic ring of WNA is highly effective regarding the stability of the triplex DNA. In this study, we designed new WNA analogs having the p-aminobenzene as an aromatic ring, which were synthesized via the Curtius–Yamada rearrangement. Based on the evaluation of the triplex formation with p-amino-WNA-TFO, it has been shown that the amino group may produce a non-selective interaction with the phosphate backbone of the target duplexes. These results indicate that the amino modification is useful to overcome the sequence-dependence of the TFO containing WNA analogs.

A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER

The promoter of human telomerase reverse transcriptase (hTERT) gene, in the region from − 1000 to + 1, contains two homopurine–homopyrimidine sequences (− 835/− 814 and − 108/− 90), that can be considered as potential targets to triple helix forming oligonucleotides (TFOs) for applying antigene strategy. We have chosen the sequence (− 108/− 90) on the basis of its unfavorable chromatin organization, evaluated by theoretical nucleosome positioning and nuclease hypersensitive sites mapping. On this sequence, anti-parallel triplex with satisfactory thermodynamic stability is formed by two TFOs, having different lengths. Triplex stability is significantly increased by specific interactions with the perylene derivative N,N′-bis[3,3′-(dimethylamino) propylamine]-3,4,9,10-perylenetetracarboxylic diimide (DAPER). Since DAPER is a symmetric molecule, the induced Circular Dichroism (CD) spectra in the range 400–600 nm allows us to obtain information on drug binding to triplex and duplex DNA. The drug-induced ellipticity is significantly higher in the case of triplex with respect to duplex and, surprisingly, it increases at decreasing of DNA. A model is proposed where self-stacked DAPER binds to triplex or to duplex narrow grooves.

New insights into DNA triplexes: residual twist and radial difference as measures of base triplet non-isomorphism and their implication to sequence-dependent non-uniform DNA triplex.

DNA triplexes are formed by both isomorphic (structurally alike) and non-isomorphic (structurally dissimilar) base triplets. It is espoused here that (i) the base triplet non-isomorphism may be articulated in structural terms by a residual twist (Δt°), the angle formed by line joining the C1′…C1′ atoms of the adjacent Hoogsteen or reverse Hoogsteen (RH) base pairs and the difference in base triplet radius (Δr Å), and (ii) their influence on DNA triplex is largely mechanistic, leading to the prediction of a high (t + Δt)° and low (t − Δt)° twist at the successive steps of Hoogsteen or RH duplex of a parallel or antiparallel triplex. Efficacy of this concept is corroborated by molecular dynamics (MD) simulation of an antiparallel DNA triplex comprising alternating non-isomorphic G*GC and T*AT triplets. Conformational changes necessitated by base triplet non-isomorphism are found to induce an alternating (i) high anti and anti glycosyl and (ii) BII and an unusual BIII conformation resulting in a zigzag backbone for the RH strand. Thus, base triplet non-isomorphism causes DNA triplexes into exhibiting sequence-dependent non-uniform conformation. Such structural variations may be relevant in deciphering the specificity of interaction with DNA triplex binding proteins. Seemingly then, residual twist (Δt°) and radial difference (Δr Å) suffice as indices to define and monitor the effect of base triplet non-isomorphism in nucleic acid triplexes.

Theoretical Study of the Guanine → 6-Thioguanine Substitution in Duplexes, Triplexes, and Tetraplexes

Molecular dynamics and thermodynamic integration calculations have been carried out on a set of G-rich single-strand, duplex, triplex, and quadruplex DNAs to study the structural and stability changes connected with the guanine --> 6-thioguanine (G --> S) mutation. The presence of 6-thioguanine leads to a shift of the geometry from the B/A intermediate to the pure B-form in duplex DNA. The G --> S mutation does not largely affect the structure of the antiparallel triplex when it is located at the reverse-Hoogsteen position, but leads to a non-negligible local distortion in the structure when it is located at the Watson-Crick position. The G --> S mutation leads to destabilization of all studied structures: the lowest effect has been observed for the G --> S mutation in the reverse-Hoogsteen strand of the triplex, a medium effect has been observed in the Watson-Crick strand of the triplex and duplex, and the highest influence of the G -->S mutation has been found for the quadruplex structures.