Hybrid Triplex Research Articles

Integration of DNA-RNA-triplex-based regulation of transcription into molecular logic gates.

In recent years, increasing numbers of noncoding RNA molecules were identified as possible components of endogenous DNA-RNA hybrid triplexes involved in gene regulation. Triplexes are potentially involved in complex molecular signaling networks that, if understood, would allow the engineering of biological computing components. Here, by making use of the enhancing and inhibiting effects of such triplexes, we demonstrate invitro the construction of triplex-based molecular gates: 'exclusive OR' (XOR), 'exclusive NOT-OR' (XNOR), and a threshold gate, via transcription of a fluorogenic RNA aptamer. Precise modulation was displayed by the biomolecular-integrated systems over a wide interval of transcriptional outputs, ranging from drastic inhibition to significant enhancement. The present contribution represents a first example of molecular gates developed using DNA-RNA triplex nanostructures.

Atypical structures of GAA/TTC trinucleotide repeats underlying Friedreich's ataxia: DNA triplexes and RNA/DNA hybrids.

Expansion of the GAA/TTC repeats in the first intron of the FXN gene causes Friedreich’s ataxia. Non-canonical structures are linked to this expansion. DNA triplexes and R-loops are believed to arrest transcription, which results in frataxin deficiency and eventual neurodegeneration. We present a systematic in silico characterization of the possible DNA triplexes that could be assembled with GAA and TTC strands; the two hybrid duplexes [r(GAA):d(TTC) and d(GAA):r(UUC)] in an R-loop; and three hybrid triplexes that could form during bidirectional transcription when the non-template DNA strand bonds with the hybrid duplex (collapsed R-loops, where the two DNA strands remain antiparallel). For both Y·R:Y and R·R:Y DNA triplexes, the parallel third strand orientation is more stable; both parallel and antiparallel protonated d(GA+A)·d(GAA):d(TTC) triplexes are stable. Apparent contradictions in the literature about the R·R:Y triplex stability is probably due to lack of molecular resolution, since shifting the third strand by a single nucleotide alters the stability ranking. In the collapsed R-loops, antiparallel d(TTC+)·d(GAA):r(UUC) is unstable, while parallel d(GAA)·r(GAA):d(TTC) and d(GA+A)·r(GAA):d(TTC) are stable. In addition to providing new structural perspectives for specific therapeutic aims, our results contribute to a systematic structural basis for the emerging field of quantitative R-loop biology.

Ion Mobility-Mass Spectrometry Reveals Details of Formation and Structure for GAA·TCC DNA and RNA Triplexes.

DNA and RNA triplexes are thought to play key roles in a range of cellular processes such as gene regulation and epigenetic remodeling and have been implicated in human disease such as Friedreich's ataxia. In this work, ion mobility-mass spectrometry (IM-MS) is used with supporting UV-visible spectroscopy to investigate DNA triplex assembly, considering stability and specificity, for GAA·TTC oligonucleotide sequences of relevance to Friedreich's ataxia. We demonstrate that, contrary to other examples, parallel triplex structures are favored for these sequences and that stability is enhanced by increasing oligonucleotide length and decreasing pH. We also provide evidence for the self-association of these triplexes, consistent with a proposed model of higher order DNA structures formed in Friedreich's ataxia. By comparing triplex assembly using DNA- and RNA-based triplex-forming oligonucleotides, we demonstrate more favorable formation of RNA triplexes, suggesting a role for their formation in vivo. Finally, we interrogate the binding properties of netropsin, a known polyamide triplex destabilizer, with RNA-DNA hybrid triplexes, where preference for duplex binding is evident. We show that IM-MS is able to report on relevant solution-phase populations of triplex DNA structures, thereby further highlighting the utility of this technology in structural biology. Our data therefore provides new insights into the possible DNA and RNA assemblies that may form as a result of GAA triplet repeats. Graphical Abstract ᅟ.

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.

Bifacial peptide nucleic acid as an allosteric switch for aptamer and ribozyme function.

We demonstrate herein that bifacial peptide nucleic acid (bPNA) hybrid triplexes functionally substitute for duplex DNA or RNA. Structure-function loss in three non-coding nucleic acids was inflicted by replacement of a duplex stem with unstructured oligo-T/U strands, which are bPNA binding sites. Functional rescue was observed on refolding of the oligo-T/U strands into bPNA triplex hybrid stems. Bifacial PNA binding was thus used to allosterically switch-on protein and small-molecule binding in DNA and RNA aptamers, as well as catalytic bond scission in a ribozyme. Duplex stems that support the catalytic site of a minimal type I hammerhead ribozyme were replaced with oligo-U loops, severely crippling or ablating the native RNA splicing function. Refolding of the U-loops into bPNA triplex stems completely restored splicing function in the hybrid system. These studies indicate that bPNA may have general utility as an allosteric trigger for a wide range of functions in non-coding nucleic acids.

Sequence-Specific Targeting of RNA with an Oligonucleotide−Neomycin Conjugate

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.

Oxazine 170 Induces DNA:RNA:DNA Triplex Formation

The oxazine dye (Oxazine 170) was found to induce formation of a hybrid triplex structure, poly rA:(poly dT)(2), under solution conditions in which the triplex would not otherwise form. Formation of the hybrid triplex is driven by the structural selective binding of oxazine 170 to poly rA:(poly dT)(2). Oxazine 170 serves as a lead compound for the design of new compounds that can modulate triplex formation.

Neomycin-induced hybrid triplex formation.

Neomycin-induced hybrid triplex formation.

Structure and affinity of DNA binding peptides

Artificial peptides designed to form alpha-helical, beta-turn, antiparallel beta-sheet and beta-hairpin structures which are among the motifs most frequently found in natural DNA/RNA binding proteins were synthesized and their characteristic features were examined in the presence or absence of double or triple stranded DNA by means of UV melting experiments, CD spectra, SPR measurements. It was revealed that amphiphilic character arising from the specific secondary structures and positive charge in the hydrophobic face of peptides played an important role in the interaction with DNA, and that hybrid duplex and triplex were intensively stabilized by the cationic amphiphilic peptides. It was also found that these peptides could protect dsDNA against DNase 1 digestion. These results indicate that structurally designed amphiphilic peptides synthesized in the present study can be powerful tools for antisense and antigene strategies.

Structural Properties of Hybrid Triplex of Polycation Deoxyribonucleic S-methylthiourea (DNmt) Strands with a Complementary DNA Strand, Probed by Nanosecond Molecular Dynamics

The replacement of phosphodiester linkages of the polyanion DNA with S-methylthiourea linkers provides the polycation deoxyribonucleic S-methylthiourea (DNmt). Molecular dynamics studies to 1,220 ps of the hybrid triplex formed from octameric DNmt strands d(Tmt)8 with a complementary DNA oligomer strand d(Ap)8 have been carried out with explicit water solvent and Na counterions under periodic boundary conditions using the CHARMM force field and the Ewald summation method. The Watson-Crick and Hoogsteen hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for triplex structures throughout the simulation. The duplex portion of the triplex structure equilibrated at a B-DNA conformation in terms of the helical rise and other helical parameters. The dynamic structures of the DNmt·DNA·DNmt triplex were determined by examining histograms from the last 800 ps of the dynamics run. These included the hydrogen-bonding pattern (sequence recognition), three-centered bifurcating occurrences, minor groove width variations, and bending of tracts for the hybrid triplex structures. Together with the Watson-Crick hydrogen-bondings, the strong Hoogsteen hydrogen-bondings, the partially maintained three-centered bifurcatings in the Watson-Crick pair, and the medium-strength three-centered bifurcatings in the Hoogsteen pair suggest that the hybrid triplex is energetically favorable as compared to a duplex with similar base stacking, van der Waals interactions, and helical parameters. This is in agreement with our previously reported thermody- namic study, in which only triplex structures were observed in solution. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 20° for the Watson-Crick portion of the averaged structure. Propeller twist (associated with three-centered hydrogen-bonding) up to −30°, native to DNA AT base pairing, was also observed for the triplex structure. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3′-endo domain and C2′-endo domain for the DNmt strand. Water spines are observed in both minor and major grooves throughout the dynamics run. The molecular dynamics simulations of the structural properties of DNmt·DNA·DNmt hybrid triplex is compared to the DNG·DNA·DNG hybrid triplex (In DNG the -O-(PO2-)-O- linkers in DNA is replaced by -NH-C(=N2)-NH-).

A method for radioprobing DNA structures using Auger electrons

Purpose : To present a new method for radioprobing a DNA triple helix structure by Auger electrons emitted in the decay of 125 I using theoretical/computational approaches. Materials and methods : A Monte Carlo track structure method was used to simulate the damage to a triplex resulting from Auger electrons emitted in the decay of an incorporated 125 I atom in plasmid DNA. Comparison of the theoretical frequency distributions of single-strand breaks induced on the Pu and Py strands with the experimental data and a knowledge of the distances from the strand breaks to the iodine provide information on the structures otherwise difficult to obtain with X-ray crystallography. Results : In comparing theoretical frequency distributions of singlestrand breaks with the experimental data it is found that the results are very sensitive to the conformation of the triplex model used. It is found that the best fit to the experimental data results from using a hybrid triplex model, in which the base-step geometry is A-like, while the sugar puckers adopt the B-like C2'- endo conformation. Conclusions : The approach and technique presented here represent a valuable new addition to the methods available for DNA structure determination since they provide information on medium-range structure otherwize difficult to obtain in the absence of X-ray crystallography. It is concluded that currently accepted models for triplex structure are not optimal, and a modified structure is proposed that fits the radioprobing results better, while maintaining agreement with the fibre diffraction and NMR data. Although the method has proved to be very useful for scoring alternative trial solutions, further studies combining experimental data from multiple iodine positions with track structure modelling are required for directing structural optimization.

Conformational and Thermodynamic Properties of Parallel Intramolecular Triple Helices Containing a DNA, RNA, or 2‘-OMeDNA Third Strand

The thermodynamic stability and solution conformational properties of three intramolecular triple helices based on the sequence AGAAGA-x-TCTTCT-x-TCTTCT (x is a non-nucleotide linker) comprising a DNA duplex and DNA, RNA, or 2‘-OMeDNA third strands have been compared. The most stable triple helix contains the 2‘-OMe third strand, followed by the triplex containing RNA in the third strand. Comparison of the NMR spectroscopic data for the RNA hybrid triplex with those of the all-DNA triplex shows that the duplex parts of the structure are very similar; the major difference is that the RNA strand is characterized by C3‘-endo sugars (except the two terminal residues). In the all-DNA triplex γ has a substantial fraction of the trans rotamer for both of the internal adenine residues (A3 and A4), whereas in the free duplex γ is g+ for these residues. In the RNA-containing triplex, only A3 shows the presence of γ(t), and in the 2‘-OMe state, both A3 and A4 are γ(g+). In addition, the 2‘-OMe triplex shows conforma...

Evidence from CD spectra and melting temperatures for stable Hoogsteen-paired oligomer duplexes derived from DNA and hybrid triplexes.

The pyr*pur.pyr type of nucleic acid triplex has a purine strand that is Hoogsteen-paired with a parallel pyrimidine strand (pyr*pur pair) and that is Watson-Crick-paired with an antiparallel pyrimidine strand (pur.pyr pair). In most cases, the Watson-Crick pair is more stable than the Hoogsteen pair, although stable formation of DNA Hoogsteen-paired duplexes has been reported. Using oligomer triplexes of repeating d(AG)12 and d(CT)12 or r(CU)12 sequences that were 24 nt long, we found that hybrid RNA*DNA as well as DNA*DNA Hoogsteen-paired strands of triplexes can be more stable than the Watson-Crick-paired strands at low pH. The structures and relative stabilities of these duplexes and triplexes were evaluated by circular dichroism (CD) spectroscopy and UV absorption melting studies of triplexes as a function of pH. The CD contributions of Hoogsteen-paired RNA*DNA and DNA*DNA duplexes were found to dominate the CD spectra of the corresponding pyr*pur.pyr triplexes.

Nanosecond Molecular Dynamics of Hybrid Triplex and Duplex of Polycation Deoxyribonucleic Guanidine Strands with a Complimentary DNA Strand

Replacement of the phosphodiester linkages of the polyanion DNA with guanidinium linkers provides the polycation deoxyribonucleic guanidine (DNG). Molecular dynamics studies to 1120 ps of the hybrid triplex and 1420 ps of the hybrid duplex formed from octameric DNG strands d(Tg)8 with a complementary DNA oligomer strand d(Ap)8 have been carried out with water explicitly as solvent and Na+Cl- as counterions under periodic boundary conditions using the CHARMM force field and Ewald summation method. The Watson−Crick and Hoogstein hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for both hybrid duplex and triplex structures throughout the simulation. And for the hybrid duplex and triplex structures, both 5‘ and 3‘ ends were only partially frayed. In agreement with our previous thermodynamic study the hybrid triplex is more favorable than the hybrid duplex in the simulations. This conclusion is reached by the comparison of the extent of 5‘ end fraying and the bifurcating properties of the two systems. The structural histories of the DNG·DNA·DNG triplex and DNG·DNA duplex were determined by examining histograms from the last 1400 ps (duplex) and 1100 ps (triplex) of the dynamics run. These include the hydrogen-bonding pattern (sequence recognition) and base pair opening occurrences, minor groove width narrowing, and tract bending for the hybrid duplex and triplex structures. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 30° for the averaged structures of both hybrid duplex and Watson−Crick portion of the hybrid triplex. Propeller twist (associated with three centered hydrogen bonding) up to −30°, native to DNA AT base pairing, was also observed for the duplex structure. The helical rise and twist parameters of the duplex structure and the duplex portion of the triplex structure assume a more B-DNA like conformation. However, the sugar pucker and other helical, axis, and base pair parameters of the two structures equilibrate at a A and B complex conformation, from base to base and base pair to base pair, which suggests a more A-DNA like overall conformation. Water spines are observed during the dynamics runs for both hybrid duplex and triplex structures in both minor and major grooves. The counterions remain in their positions through the dynamics run for the triplex system.

Modulation of nucleic acid structure by ligand binding: induction of a DNA·RNA·DNA hybrid triplex by DAPI intercalation

The aromatic diamidine, DAPI (4′,6-diamidino-2-phenylindole), is used as an important biological and cytological tool since it forms highly fluorescent complexes with nucleic acid duplexes via minor groove-directed/intercalative modes of interaction. In this study, we find that DAPI binding can induce the formation of an RNA-DNA hybrid triplex that would not otherwise form. More specifically, through application of a broad range of spectroscopic, viscometric, and molecular modeling techniques, we demonstrate that DAPI intercalation induces the formation of the poly(dT)·poly(rA)·poly(dT) hybrid triple helix, a structure which does not form in the absence of the ligand. Using UV mixing studies, we demonstrate that, in the presence of DAPI, the poly(rA)·poly(dT) duplex and the poly(dT) single strand form a 1:1 complex (a triplex) that does not form in the absence of DAPI. Through temperature-dependent absorbance measurements, we show that the poly(dT)·poly(rA)·poly(dT) triplex melts via two distinct transitions: initial conversion of the triplex to the duplex state, with the DAPI remaining bound, followed by denaturation of the duplex-DAPI complex to its component single strands and free DAPI. Using optical melting profiles, we show that DAPI binding enhances the thermal stability of the poly(dT)·poly(rA)·poly(dT) triplex, an observation consistent with the preferential binding of the ligand to the triplex versus the duplex and single-stranded states. Our differential scanning calorimetric measurements reveal melting of the DAPI-saturated poly(dT)·poly(rA)·poly(dT) triplex to be associated with a lower enthalpy but greater cooperativity than melting of the corresponding DAPI-saturated poly(rA)·poly(dT) duplex. Our flow linear dichroism and viscometric data are consistent with an intercalative mode of binding when DAPI interacts with both the poly(dT)·poly(rA)·poly(dT) triplex and the poly(rA)·poly(dT) duplex. Finally, computer modeling studies suggest that a combination of both stacking and electrostatic interactions between the intercalated ligand and the host nucleic acid play important roles in the DAPI-induced stabilization of the poly(dT)·poly(rA)·poly(dT) triplex. In the aggregate, our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, antiviral, and diagnostic strategies.

Helical periodicity of GA-alternating triple-stranded DNA.

Homopurine-homopyrimidine tracts (18 or 28 bp) containing predominantly GA-alternating sequences were inserted between two bent DNA loci composed of six (dA)6.(dT)6 repeats. For each homopurine tract, the DNA length between the bent DNA loci was varied by one base pair over a full helical turn. The two series of bent DNA fragments were electrophoresed in 5% polyacrylamide gel to measure the gel mobilities, which reflected the overall extent of DNA bending of each DNA fragment. By comparing the gel mobilities between the two series of bent DNA fragments, the helical periodicity of the GA-alternating duplex was determined to be 10.4 bp/turn. The two series of bent DNA fragments were also electrophoresed upon formation of the intermolecular triplex at the homopurine.homopyrimidine tracts. Comparison of the gel mobilities between the two series of DNA fragments showed that the helical periodicity of triplexed DNA of GA-alternating sequence was 11.2 bp/turn. Triplexed DNA with homopurine and homopyrimidine oligodeoxyribonucleotides had the same helical periodicity, while hybrid triplex DNA associated with homopyrimidine oligoribonucleotide had a slightly more wound structure with 11.1 bp/turn.