Binding of oligonucleotides to a viral hairpin forming RNA triplexes with parallel G*G*C triplets

Infrared and UV spectroscopies have been used to study the assembly of a hairpin nucleotide sequence (nucleotides 3–30) of the 5′ non-coding region of the hepatitis C virus RNA (5′-GGCGGGGAUUAUCCCCGCUGUGAGGCGG-3′) with a RNA 20mer ligand (5′-CCGCCUCACAAAGGUGGGGU-3′) in the presence of magnesium ion and spermidine. The resulting complex involves two helical structural domains: the first one is an intermolecular duplex stem at the bottom of the target hairpin and the second one is a parallel triplex generated by the intramolecular hairpin duplex and the ligand. Infrared spectroscopy shows that N-type sugars are exclusively present in the complex. This is the first case of formation of a RNA parallel triplex with purine motif and shows that this type of targeting RNA strands to viral RNA duplexes can be used as an alternative to antisense oligonucleotides or ribozymes.     

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.     

Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices

A total of 16 oligodeoxyribonucleotides of general sequence 5′-TCTTCTZTCTTTCT-3′, where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5′-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3′, where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an ‘interrupting’ T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4°C higher than that of the triplex with 5′-TCTTCTGTCTTTCT-3′ as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.     

Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides

The interactions of pyrimidine deoxyribo- or 2′-O-methylribo-psoralen-conjugated, triplex-forming oligonucleotides, psTFOs, with a 17-bp env-DNA whose purine tract is 5′-AGAGAGAAAAAAGAG-3′, or an 18-bp gag-DNA whose purine tract is 5′-AGG GGGAAAGAAAAAA-3′, were studied over the pH range 6.0–7.5. The stability of the triplex formed by a deoxy-env-psTFO containing 5-methylcytosines and thymines decreased with increasing pH (T_m = 56°C at pH 6.0; 27°C at pH 7.5). Replacement of 5-methylcytosines with 8-oxo-adenines reduced the pH dependence, but lowered triplex stability. A 2′-O-methyl-env-psTFO containing uracil and cytosine did not form a triplex at pH 7.5. Surprisingly, replacement of the cytosines in this oligomer with 5-methylcytosines dramatically increased triplex stability (T_m = 25°C at pH 7.5), and even greater stability was achieved by selective replacement of uracils with thymines (T_m = 37°C at pH 7.5). Substitution of the contiguous 5-methylcytosines of the deoxy-gag-psTFO with 8-oxo-adenines significantly reduced pH dependence and increased triplex stability. In contrast to the behavior of env-specific TFOs, triplexes formed by 2′-O-methyl-gag-psTFOs did not show enhanced stability. Replacement of the 3′-terminal phosphodiester of the TFO with a methylphosphonate group significantly increased the resistance of both deoxy- and 2′-O-methyl-TFOs to degradation by 3′-exonucleases, while maintaining triplex stability.     

Triplex formation with 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) having C3'-endo conformation at physiological pH

Antigenes, which are substances that inhibit gene expression by binding to double-stranded DNA (dsDNA) in a sequence-specific manner, are currently sought for the treatment of various gene-related diseases. As such antigenes, we developed new nuclease-resistant oligopyrimidine nucleotides that are partially modified with 2′-O,4′-C-ethylene nucleic acids (ENA), which are constrained in the C3′-endo conformation and can form a triplex with dsDNA at physiological pH. It was found that these oligonucleotides formed triplexes similarly to those partially modified with 2′-O,4′-C-methylene nucleic acids (2′,4′-BNA or LNA), as determined by UV melting analyses, electromobility shift assays, CD spectral analyses and restriction enzyme inhibition assays. In our studies, oligonucleotides fully modified with ENA have δ torsion angle values that are marginally higher than those of 2′,4′-BNA/LNA. ENA oligonucleotides present in 10-fold the amount of dsDNA were found to be favorable in forming triplexes. These results provide useful information for the future design of triplex-forming oligonucleotides fully modified with such nucleic acids constrained in the C3′-endo conformation considering that oligonucleotides fully modified with 2′,4′-BNA/LNA do not form triplexes.     

RNA cleavage products generated by antisense oligonucleotides and siRNAs are processed by the RNA surveillance machinery

DNA-based antisense oligonucleotides (ASOs) elicit cleavage of the targeted RNA by the endoribonuclease RNase H1, whereas siRNAs mediate cleavage through the RNAi pathway. To determine the fates of the cleaved RNA in cells, we lowered the levels of the factors involved in RNA surveillance prior to treating cells with ASOs or siRNA and analyzed cleavage products by RACE. The cytoplasmic 5′ to 3′ exoribonuclease XRN1 was responsible for the degradation of the downstream cleavage products generated by ASOs or siRNA targeting mRNAs. In contrast, downstream cleavage products generated by ASOs targeting nuclear long non-coding RNA Malat 1 and pre-mRNA were degraded by nuclear XRN2. The downstream cleavage products did not appear to be degraded in the 3′ to 5′ direction as the majority of these products contained intact poly(A) tails and were bound by the poly(A) binding protein. The upstream cleavage products of Malat1 were degraded in the 3′ to 5′ direction by the exosome complex containing the nuclear exoribonuclease Dis3. The exosome complex containing Dis3 or cytoplasmic Dis3L1 degraded mRNA upstream cleavage products, which were not bound by the 5′-cap binding complex and, consequently, were susceptible to degradation in the 5′ to 3′ direction by the XRN exoribonucleases.     

Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 Å decrease in the separation between phosphorothioate and 2′F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR–tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, ³¹P–¹⁹F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA–peptide complex.     

Molecular recognition of poly(A) by small ligands: an alternative method of analysis reveals nanomolar, cooperative and shape-selective binding

A few drug-like molecules have recently been found to bind poly(A) and induce a stable secondary structure (T_m ≈ 60°C), even though this RNA homopolymer is single-stranded in the absence of a ligand. Here, we report results from experiments specifically designed to explore the association of small molecules with poly(A). We demonstrate that coralyne, the first small molecule discovered to bind poly(dA), binds with unexpectedly high affinity (K_a >10⁷ M⁻¹), and that the crescent shape of coralyne appears necessary for poly(A) binding. We also show that the binding of similar ligands to poly(A) can be highly cooperative. For one particular ligand, at least six ligand molecules are required to stabilize the poly(A) self-structure at room temperature. This highly cooperative binding produces very sharp transitions between unstructured and structured poly(A) as a function of ligand concentration. Given the fact that junctions between Watson–Crick and A·A duplexes are tolerated, we propose that poly(A) sequence elements and appropriate ligands could be used to reversibly drive transitions in DNA and RNA-based molecular structures by simply diluting/concentrating a sample about the poly(A)-ligand ‘critical concentration’. The ligands described here may also find biological or medicinal applications, owing to the 3′-polyadenylation of mRNA in living cells.     

Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°₃₇, and T_M for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5′ or 3′ position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5′ or 3′ side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.     

Protein-free parallel triple-stranded DNA complex formation

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO₄-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.     

Recombination R-triplex: H-bonds contribution to stability as revealed with minor base substitutions for adenine

Several cellular processes involve alignment of three nucleic acids strands, in which the third strand (DNA or RNA) is identical and in a parallel orientation to one of the DNA duplex strands. Earlier, using 2-aminopurine as a fluorescent reporter base, we demonstrated that a self-folding oligonucleotide forms a recombination-like structure consistent with the R-triplex. Here, we extended this approach, placing the reporter 2-aminopurine either in the 5′- or 3′-strand. We obtained direct evidence that the 3′-strand forms a stable duplex with the complementary central strand, while the 5′-strand participates in non-Watson–Crick interactions. Substituting 2,6-diaminopurine or 7-deazaadenine for adenine, we tested and confirmed the proposed hydrogen bonding scheme of the A*(T·A) R-type triplet. The adenine substitutions expected to provide additional H-bonds led to triplex structures with increased stability, whereas the substitutions consistent with a decrease in the number of H-bonds destabilized the triplex. The triplex formation enthalpies and free energies exhibited linear dependences on the number of H-bonds predicted from the A*(T·A) triplet scheme. The enthalpy of the 10 nt long intramolecular triplex of −100 kJ·mol⁻¹ demonstrates that the R-triplex is relatively unstable and thus an ideal candidate for a transient intermediate in homologous recombination, t-loop formation at the mammalian telomere ends, and short RNA invasion into a duplex. On the other hand, the impact of a single H-bond, 18 kJ·mol⁻¹, is high compared with the overall triplex formation enthalpy. The observed energy advantage of a ‘correct’ base in the third strand opposite the Watson–Crick base pair may be a powerful mechanism for securing selectivity of recognition between the single strand and the duplex.     

A directional nucleation-zipping mechanism for triple helix formation

A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis. Replacement of a T·A base pair by a C·G pair at either the 5′ or the 3′ end of the target sequence allowed us to assess mismatch effects and to delineate the mechanism of triple helix formation. Our data show that the association rate constant is governed by the sequence of base triplets on the 5′ side of the triplex (referred to as the 5′ side of the target oligopurine strand) and provides evidence that the reaction pathway for triple helix formation in the pyrimidine motif proceeds from the 5′ end to the 3′ end of the triplex according to the nucleation-zipping model. It seems that this is a general feature for all triple helices formation, probably due to the right-handedness of the DNA double helix that provides a stronger base stacking at the 5′ than at the 3′ duplex–triplex junction. Understanding the mechanism of triple helix formation is not only of fundamental interest, but may also help in designing better triple helix-forming oligonucleotides for gene targeting and control of gene expression.     

Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand

We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (K(d) approximately 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.     

Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5′-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life ~10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations ~0.1 μM for acridine-conjugated TFO/LNA (or ~2 μM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated.     

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T·AT triplets are unstable and melt below 30°C. In contrast, triplets with repeats of TTC, TCC and CTCT melt at 67, 72 and 76°C, respectively. The most stable complex is generated by the sequence containing alternating C⁺·GC and T·AT triplets. All four triplexes are stabilised by increasing the ionic strength or by the addition of magnesium, although triplexes with a higher proportion of C⁺·GC triplets are much less sensitive to changes in the ionic conditions. The enthalpies of formation of these triplexes were estimated by examining the concentration dependence of the melting profiles and show that, in the presence of 200 mM sodium at pH 5.0, each C⁺·GC triplet contributes about 30 kJ mol^–1, while each T·AT contributes only 11 kJ mol^–1. Kinetic experiments with these oligonucleotides show that in 200 mM sodium (pH 5.0) repeats of TCC and TTC have half-lives of ~20 min, while the triplex with alternating C⁺·GC and T·AT triplets has a half-life of ~3 days. In contrast, the dissociation kinetics of the triplex containing only T·AT are too fast to measure.     

Relative stability of triplexes containing different numbers of T.AT and C+.GC triplets.

We have used DNase I footprinting to compare the stability of parallel triple helices containing different numbers of T.AT and C+. GC triplets. We have targeted a fragment containing the 17mer sequence 5'-AGGAAGAGAAAAAAGAA with the 9mer oligonucleotides 5'-TCCTTCTCT, 5'-TTCTCTTTT and 5'-TTTTTTCTT, which form triplexes at the 5'-end, centre and 3'-end of the target site respectively. Quantitative DNase I footprinting has shown that at pH 5.0 the dissociation constants of these oligonucleotides are 0.13, 4.7 and >30 microM respectively, revealing that increasing the proportion of C+.GC triplets increases triplex stability. The results suggest that the positive charge on the protonated cytosine contributes to triplex stability, either by a favourable interaction with the stacked pisystem or by screening the charge on the phosphate groups. In the presence of a naphthylquinoline triplex binding ligand all three oligonucleotides bind with similar affinities. At pH 6.0 these triplexes only form in the presence of the triplex binding ligand, while at pH 7.5 footprints are only seen with the oligonucleotide which generates the fewest number of C+.GC triplets (TTTTTTCTT) in the presence of the ligand.     

The effect of amino groups on the stability of DNA duplexes and triplexes based on purines derived from inosine

The effect of amino groups attached at positions 2 and 8 of the hypoxanthine moiety in the structure, reactivity and stability of DNA duplexes and triplexes is studied by means of quantum mechanical calculations, as well as extended molecular dynamics (MD) and thermodynamic integration (MD/TI) simulations. Theoretical estimates of the change in stability related to 2′-deoxyguanosine (G) → 2′-deoxyinosine (I) → 8-amino-2′-deoxyinosine (8AI) mutations have been experimentally verified, after synthesis of the corresponding compounds. An amino group placed at position 2 stabilizes the duplex, as expected, and surprisingly also the triplex. The presence of an amino group at position 8 of the hypoxanthine moiety stabilizes the triplex but, surprisingly, destabilizes the duplex. The subtle electronic redistribution occurring upon the introduction of an amino group on the purine seems to be responsible for this surprising behavior. Interesting ‘universal base’ properties are found for 8AI.     

Selective Preference of Parallel DNA Triplexes Is Due to the Disruption of Hoogsteen Hydrogen Bonds Caused by the Severe Nonisostericity between the G*GC and T*AT Triplets

Implications of DNA, RNA and RNA.DNA hybrid triplexes in diverse biological functions, diseases and therapeutic applications call for a thorough understanding of their structure-function relationships. Despite exhaustive studies mechanistic rationale for the discriminatory preference of parallel DNA triplexes with G_*GC & T_*AT triplets still remains elusive. Here, we show that the highest nonisostericity between the G_*GC & T_*AT triplets imposes extensive stereochemical rearrangements contributing to context dependent triplex destabilisation through selective disruption of Hoogsteen scheme of hydrogen bonds. MD simulations of nineteen DNA triplexes with an assortment of sequence milieu reveal for the first time fresh insights into the nature and extent of destabilization from a single (non-overlapping), double (overlapping) and multiple pairs of nonisosteric base triplets (NIBTs). It is found that a solitary pair of NIBTs, feasible either at a G_*GC/T_*AT or T_*AT/G_*GC triplex junction, does not impinge significantly on triplex stability. But two overlapping pairs of NIBTs resulting from either a T_*AT or a G_*GC interruption disrupt Hoogsteen pair to a noncanonical mismatch destabilizing the triplex by ~10 to 14 kcal/mol, implying that their frequent incidence in multiples, especially, in short sequences could even hinder triplex formation. The results provide (i) an unambiguous and generalised mechanistic rationale for the discriminatory trait of parallel triplexes, including those studied experimentally (ii) clarity for the prevalence of antiparallel triplexes and (iii) comprehensive perspectives on the sequence dependent influence of nonisosteric base triplets useful in the rational design of TFO’s against potential triplex target sites.