Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.

A molecular anchor for stabilizing triple-helical DNA.

Molecular modeling has been used to predict that 2,6-disubstituted amidoanthraquinones, and not the 1,4 series, should preferentially interact with and stabilize triple-stranded DNA structures over duplex DNA. This is due to marked differences in the nature of chromophore-base stacking and groove accessibility for the two series. A DNA foot-printing method that monitors the extent of protection from DNase I cleavage on triplex formation has been used to examine the effects of a number of synthetic isomer compounds in the 1,4 and 2,6 series. The experimental results are in accord with the predicted behavior and confirm that the 1,4 series bind preferentially to double- rather than triple-stranded DNA, whereas the isomeric 2,6 derivatives markedly favor binding to triplex DNA.

Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts

Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.

Stabilisation of TG- and AG-containing antiparallel DNA triplexes by triplex-binding ligands

We have used DNase I footprinting to examine the interaction of several triplex-binding ligands with antiparallel TG- and AG-containing triplexes. We find that although a 17mer TG-containing oligonucleotide on its own fails to produce a footprint at concentrations as high as 30 µM, this interaction can be stabilised by several ligands. Within a series of disubstituted amidoanthraquinones we find that the 2,7- regioisomer affords the best stabilisation of this TG triplex, though the 1,8- isomer also stabilises this interaction to some extent. By contrast the 1,5- and 2,6- regioisomers show no interaction with TG triplexes. Similar studies with a 13mer AG-containing oligonucleotide show the opposite pattern of stabilisation: the 2,6- and 1,5- isomers stabilise this triplex, but the 2,7- and 1,8-compounds do not. The polycyclic compound BePI strongly stabilises TG- but not AG-containing triplexes, while a substituted naphthylquinoline interacts with both antiparallel triplex motifs.

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.

Detection of competing DNA structures by thermal gradient gel electrophoresis: from self-association to triple helix formation by (G,A)-containing oligonucleotides

Sequence-specific recognition of DNA can be achieved by triple helix-forming oligonucleotides that bind to the major groove of double-helical DNA. These oligonucleotides have been used as sequence-specific DNA ligands for various purposes, including sequence-specific gene regulation in the so-called ‘antigene strategy’. In particular, (G,A)-containing oligonucleotides can form stable triple helices under physiological conditions. However, triplex formation may be in competition with self-association of these oligonucleotides. For biological applications it would be interesting to identify the conditions under which one structure is favoured as compared to the other(s). Here we have directly studied competition between formation of a parallel (G,A) homoduplex and that of a triple helix by a 13 nt (G,A)-containing oligonucleotide. Temperature gradient gel electrophoresis allows simultaneous detection of competition between the two structures, because of their different temperature dependencies and gel electrophoretic mobilities, and characterisation of this competition.

Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Prediction of the stability of DNA triplexes.

We present rules that allow one to predict the stability of DNA pyrimidine.purine.pyrimidine (Y.R.Y) triple helices on the basis of the sequence. The rules were derived from van't Hoff analysis of 23 oligonucleotide triplexes tested at a variety of pH values. To predict the enthalpy of triplex formation (delta H degrees), a simple nearest-neighbor model was found to be sufficient. However, to accurately predict the free energy of the triplex (delta G degrees), a combination model consisting of five parameters was needed. These parameters were (i) the delta G degrees for helix initiation, (ii) the delta G degrees for adding a T-A.T triple, (iii) the delta G degrees for adding a C(+)-G.C triple, (iv) the penalty for adjacent C bases, and (v) the pH dependence of the C(+)-G.C triple's stability. The fitted parameters are highly consistent with thermodynamic data from the basis set, generally predicting both delta H degrees and delta G degrees to within the experimental error. Examination of the parameters points out several interesting features. The combination model predicts that C(+) -G.C. triples are much more stabilizing than T-A.T triples below pH 7.0 and that the stability of the former increases approximately equal to 1 kcal/mol per pH unit as the pH is decreased. Surprisingly though, the most stable sequence is predicted to be a CT repeat, as adjacent C bases partially cancel the stability of one another. The parameters successfully predict tm values from other laboratories, with some interesting exceptions.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.