DNA Triple Helix Formation Research Articles

Specificity of antiparallel DNA triple helix formation.

We have used DNase I footprinting to examine the formation of antiparallel DNA triple helices on DNA fragments containing the homopurine target sites (GGA)2GGX(GGA)2GG.(CCT)2CCZ(CCT)2CC (where X.Z is each base pair in turn), with the GA- and GT-rich oligonucleotides, (GGA)2GGN(GGA)2GG and (GGT)2GGN(GGT)2GG (N = each base in turn). These were designed to form G.GC and A.AT or T.AT triplets with a central N.XZ mismatch, which should bind in an antiparallel orientation. We find that almost all combinations generate DNase I footprints at low micromolar concentrations. At each target site, the relative binding of the GA- and GT-containing oligonucleotides was not the same, suggesting that these two triplexes adopt different conformations. For a central GC base pair, the most stable complex is observed with a third strand generating a G.GC triplet as expected. A.GC is also stable, especially in the GT oligonucleotides. For a central AT base pair, all four bases form stable complexes though T.AT is favored for the GA-rich thirds strands and A.AT for the GT-rich strands. For a central CG base pair, the stable complexes are seen with third strands generating T.CG triplets, though A.CG and C.CG are stable with GT- and GA-containing oligonucleotides, respectively. C.TA is the best triplet at a central TA base pair. The third strands with central guanines avoided the formation of G.YR triplets on the fragments containing central pyrimidines, producing DNase I footprints which had slipped relative to the target site. These oligonucleotides bound at a different location, generating complexes containing 11 contiguous stable triplets at the 3'-end of the third strand. The results suggest rules for designing the best third strand oligonucleotides for targeting sequences in which homopurine tracts are interrupted by pyrimidines.

Stabilization of DNA triple-helix formation by appended cationic peptides.

We have investigated the impact of appended cationic peptides on the triplex-forming and thermal-stabilizing abilities of an oligonucleotide "third strand" using a homopurine-homopyrimidine 37-mer as the host duplex target. The family of appended cationic peptides studied here contains arginine, lysine, ornithine, and diaminobutyric acid, with the residues being linked through either their alpha-amino or side chain amino groups. On the basis of optical melting profiles, we find that peptides with different amino acid compositions, but with four positive charges in common, are able to enhance, in a similar manner, the triplex-forming capability of an oligonucleotide. We note the implications of these results for the rational design of third-strand probes.

An Unusually Stable Purine(Purine-Pyrimidine) Short Triplex.: THE THIRD STRAND STABILIZES DOUBLE-STRANDED DNA

Classical models for DNA triple helix formation assume the stabilization of these structures through the formation of Hoogsteen hydrogen bonds. This assumes that G-rich duplex DNA is more stable than triplex DNA. We report the results of co-migration assay, dimethyl sulfate footprint, and UV spectroscopic melting studies that reveal that at least in some cases of short (13-mer) purine(purine-pyrimidine) triplex the stability of double-stranded DNA is increased by the binding of the third strand. Under conditions which are usually considered as physiological (10 mM MgCl2, 150 mM Na+ or K+) and with a rate of heating/cooling of 1 degrees C/min, there is a good reversibility of the melting profiles which is consistent with a high rate of triplex formation. Other factors than Hoogsteen hydrogen bonds should therefore be involved in triplex stabilization. We suggest that oligonucleotides with similar properties could be efficient agents for artificial gene regulation.

Extension of DNA triple helix formation to a neighbouring (AT) n site

We have used DNase I footprinting to examine the formation of intermolecular triple helices at a fragment containing the target sequence A 11(AT) 6 · (AT) 6T 11, using oligonucleotides designed to form parallel T · AT and G · TA triplets. We find that, although (TG) 6 does not form a complex with (AT) 6 · (AT) 6, T 11(TG) 6 forms a stable structure producing a clear footprint which includes the (AT) 6 portion of the target site. This complex is not formed in the presence of magnesium, but can be stabilised by either manganese or a triplex-binding ligand.

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.

Direct measurement of thermodynamic and kinetic parameters of DNA triple helix formation by fluorescence spectroscopy.

Direct measurement of thermodynamic and kinetic parameters of oligonucleotide-directed DNA triple helix formation has been achieved by fluorescence spectroscopic methods. Fluorescence resonance energy transfer (FRET) was used to study the binding of an acceptor-labeled single-stranded oligonucleotide to a donor-labeled DNA duplex. Equilibrium binding constants and association rate constants for triplex formation between 5'-tetramethylrhodamine-labeled 11-mer, 13-mer, and 15-mer homopyrimidine oligonucleotides and a 5'-fluorescein-labeled, 25-bp DNA duplex containing a 15-bp homopurine site were determined by FRET measurements, and the values were in close agreement with those determined by established methods. The thermal dissociation profile of the triplex-to-duplex transition was also directly observed by FRET and was consistent with the triplex melting curves obtained by UV absorbance measurements. In addition, a homogeneous fluorescence anisotropy assay is described which enables determination of the binding constants between 5'-tetramethylrhodamine-labeled 11-mer and 13-mer homopyrimidine oligonucleotides and unlabeled 25-, 30-, and 50-bp double-stranded DNA containing a homopurine target site. These results demonstrate the utility of nonradioactive fluorescence measurements as an efficient method for studying triple helix formation under homogeneous solution conditions and highlight the uniqueness of the FRET method for obtaining equilibrium, kinetic, and thermal dissociation data in a straightforward manner.

Triple helix formation at A 8XA 8·T 8YT 8

We have examined the formation of DNA triple helices between the oligonucleotides T 8XT 8 (X = A,C,G,T) and DNA fragments containing the target sequences A 8XA 8 · T 8YT 8 (X = T,C,G; Y=A,G,C), by DNase I footprinting. We find that A 8GAg 8 · T 8CT 8 yields a footprint with T 8CT 8 and shows a weaker interaction with T 17 and T 8GT 8· A 8CA 8 · T 8GT 8 yields a footprint with T 17, and shows weaker interaction with T 8CT 8. A 8TA 8 · T 8AT 8 yields a footprint with T 8GT 8 and shows weaker interaction with T 17. Each of the successful complexes is characterised by enhanced DNase I cleavage at the 3' end of the purine strand of the target, as well as protection at the 5' end. We have been unable to form triplexes with third strands of the type A 8XA 8.

Energetics of a Stable Intramolecular DNA Triple Helix Formation

We have designed and synthesized by conventional chemical techniques a 38mer oligonucleotide consisting of a 5′d(Pu)10d(C)4d(Py)10d(T)4d(Py)103′ sequence. This oligonucleotide assumes a randomly coiled conformation at pH 12. At pH 8·0 a hairpin helix forms between its 5′ purine decamer sequence and the consecutive pyrimide decamer leaving the second pyrimidine decamer as a dangling disorder 3′ extension. On reducing the pH to 4·5 this second pyrimidine decamer folds back onto the major groove of the hairpin helix resulting in an intramolecular triple-stranded stem-loop structure. We have used a variety of biochemical (gel mobility, P1 nuclease digestion) and biophysical (ultraviolet light and circular dichroism spectroscopy, fluorimetry, microcalorimetry) techniques to characterize the different conformers, their stability and the folding pathway into an intramolecular triple helix. The thermodynamic properties of this intramolecular triple strand in 100 mM-Na+ are: tm , 71°C; ΔHvH, 119·4(±11·9) kcal mol 1; ΔHcal, 121·9 (±6·1) kcal mol-1 at pH 4·5; those of the hairpin are: tm , 63°C; ΔHvH, 71·7(±4·0) kcal mol-1; ΔHcal, 69·9(±3·5) kcal mol-1 at pH 8·0. At intermediate pH values, the triplex to coil transition breaks up into its component triplex to hairpin and hairpin to coil transitions with thermodynamic properties: tm 41°C; ΔHvH, 58·7(±4·2) kcal mol-1; ΔHcal, 39·8(±2·0) kcal mol-1; and tm , 63°C; ΔHvH, 71·7(±4·0) kcal mol-1; ΔHcal, 69·6(±3·5) kcal mol-1 at pH 6·7

DNA triple-helix formation: an approach to artificial gene repressors?

Certain sequences of double-helical DNA can be recognized and tightly bound by oligonucleotides. The effects of such triple-helical structures on DNA binding proteins have been studied. Stabilities of DNA triple-helices at or near physiological conditions are sufficient to inhibit DNA binding proteins directed to overlapping sites. Such proteins include restriction endonucleases, methylases, transcription factors, and RNA polymerases. These and other results suggest that oligonucleotide-directed triple-helix formation could provide the basis for designing artificial gene repressors. The general question of whether biological systems employ RNA molecules for recognition and regulation of double-helical DNA is discussed.

Inhibition of T7 RNA polymerase initiation by triple-helical DNA complexes: a model for artificial gene repression

An experimental approach is presented for the creation of an artificial and functional repressor/operator interaction that does not involve polypeptides. This in vitro approach confers oligonucleotide regulation upon a bacteriophage T7 RNA polymerase promoter by introducing an overlapping homopurine operator that can be recognized by oligonucleotide-directed DNA triple-helix formation. Recognition of optimized operator sequences in either of two triple-helix motifs is shown to efficiently inhibit T7 RNA polymerase transcription initiation in both a promoter- and oligonucleotide-specific manner. Inhibition due to triple helices of the pyrimidine motif is pH-dependent, as expected. Inhibition by purine motif triple helices is not pH-dependent and occurs efficiently under optimum T7 RNA polymerase transcription conditions. Repression by triple-helix formation can be observed rapidly after addition of purine motif repressor oligonucleotides, even when polymerase has been given prior access to the promoter. The mechanism of repression is shown to be occlusion of polymerase from the promoter rather than trapping of the polymerase in unproductive preinitiation or initiation complexes. In contrast to their inhibition of T7 RNA polymerase initiation, the triple-helical complexes studied here do not detectably inhibit transcription elongation.

DNA Triple-Helix Formation at Physiologic pH and Temperature

Oligonucleotides that form a triple helix with duplex DNA offer a novel way to site specifically regulate gene expression in vivo. Triple helices formed by homopyrimidine oligomers containing both cytosine and thymine are stabilized by acid pH and low temperature, and there is little information about triplex formation with these oligomers at both pH 7.5 and 37 degrees C. Therefore, we examined the effect of changing various conditions on triplex formation at pH 7.5. A 30-mer oligonucleotide (composed of T and 5-methyl C) at submicromolar concentrations formed a triplex with its target duplex at pH 7.5 and 37 degrees C. Association of the 30-mer oligomer with the duplex was slow, with complete association requiring about 1 h. At 37 degrees C, a 21-mer oligomer bound weakly to the target duplex but both a 25-mer and the 30-mer readily formed a triplex. This relationship of triplex formation with length was temperature dependent, as at 25 degrees C the 21-mer behaved similarly to the longer oligomers. Increasing spermine concentrations (from 0.2 to 1 mM) increased the amount of triplex formed. Spermine may be important only for the association of the oligomer to the duplex, since decreasing the spermine concentration after the triplex formed did not reduce the amount of triplex detected. At 1 mM spermine, formation of the triple-helical complex was very dependent on the concentration of KCl; increasing the KCl from 50 to 100 mM prevented triplex formation. However, the inhibitory effect of KCl could be abrogated by raising the spermine concentration to 2 mM. Our observations indicate that a triple helix can form under physiologic conditions but its formation is affected by several competing interactions.

Oligonucleotide-Directed DNA Triple-Helix Formation: An Approach to Artificial Repressors?

Pyrimidine oligonucleotides exhibit sequence-specific binding to purine sequences in double-helical DNA. Molecular recognition occurs through triple-helix formation, mediated by Hoogsteen hydrogen bonding of the pyrimidine strand parallel to the double-helical DNA purine strand. Oligonucleotide-directed DNA triple-helix formation has been shown to inhibit the recognition of double-helical DNA by sequence-specific binding proteins. This observation provides the potential for new approaches to both the analysis of nucleic acids, and the study of nucleic acid-protein interactions. We wish to understand molecular mechanisms whereby such complexes affect protein-DNA interactions involved in eukaryotic transcription.

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.