Triplex-forming Sequences Research Articles

Benzoquinoquinoxaline Derivatives Stabilize and Cleave H-DNA and Repress Transcription Downstream of a Triplex-forming Sequence

Oligopyrimidine*oligopurine sequences with potential to form intramolecular triple helix structures (H-DNA) have been found mainly in high eukaryote genomes. However, the natural occurrence and function of H-DNA remains elusive largely because we lack appropriate reagents to demonstrate the formation of these structures in cells. We examined whether a triple-helix specific stabilizing compound, benzoquinoquinoxaline (BQQ), and its 1,10-phenanthroline derivative can be efficiently utilized to study the formation and stabilization of an intramolecular triple-helical DNA structure in growing Escherichia coli cells and in vitro. Cell uptake of BQQ was confirmed by fluorescence microscopy. A plasmid carrying an H-DNA forming sequence upstream of a reporter gene was used to assess the effects of H-DNA formation and stabilization in growing cells. The presence of the H-DNA forming sequence dramatically repressed beta-lactamase expression, and sub-growth-inhibitory doses of BQQ caused a further 40% reduction. Most importantly, repression was dependent on the triple-helix forming sequence and correlated with the addition of BQQ. As the abundance of the H-DNA forming plasmid was not affected by the addition of BQQ, the dose-dependent reduction at the protein level observed here is likely caused by repression of transcription. Finally, the triple-helix specific interaction of BQQ with the target DNA sequence was demonstrated using a triple-helix directed cleavage assay by BQQ-1,10-phenanthroline conjugate in vitro.

Gene activation by triplex-forming oligonucleotide coupled to the activating domain of protein VP16.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chimera (TFO-P) can specifically recognise its DNA target at physiological salt and pH conditions. Bound to the double-stranded target DNA in a promoter region, the TFO-P is able to activate gene expression. Our results suggest that this type of molecule may prove useful in the design of new tools for artificial modulation of gene expression.

Overcoming a barrier for DNA polymerization in triplex-forming sequences.

Folded structures in the DNA template, such as hairpins and multi-stranded structures, often serve as pause and arrest sites for DNA polymerases. DNA polymerization is particularly difficult on mirror-repeated homopurine.homopyrimidine templates where triple-stranded (triplex) structures may form between the nascent and folded template strands. In order to use a linear PCR amplification approach for the structural analysis of DNA in mirror-repeated sequences we modified a conventional protocol. The barrier for DNA synthesis can be eliminated using an oligonucleotide that hybridizes with the template to prevent its folding and is subsequently displaced by the progressing polymerase. The described approach is potentially useful for sequencing and analysis of chemical adducts and point mutations in a variety of sequences prone to the formation of folded structures, such as long hairpins and quadruplexes.

Overcoming a barrier for DNA polymerization in triplex-forming sequences

Folded structures in the DNA template, such as hairpins and multi-stranded structures, often serve as pause and arrest sites for DNA polymerases. DNA polymerization is particularly difficult on mirror-repeated homopurine.homopyrimidine templates where triple-stranded (triplex) structures may form between the nascent and folded template strands. In order to use a linear PCR amplification approach for the structural analysis of DNA in mirror-repeated sequences we modified a conventional protocol. The barrier for DNA synthesis can be eliminated using an oligonucleotide that hybridizes with the template to prevent its folding and is subsequently displaced by the progressing polymerase. The described approach is potentially useful for sequencing and analysis of chemical adducts and point mutations in a variety of sequences prone to the formation of folded structures, such as long hairpins and quadruplexes.

Solution conformation of a parallel DNA triple helix with 5' and 3' triplex-duplex junctions.

Polypurine x polypyrimidine sequences of DNA can form parallel triple helices via Hoogsteen hydrogen bonds with a third DNA strand that is complementary to the purine strand. The triplex prevents transcription and could therefore potentially be used to regulate specific genes. The determination of the structures of triplex-duplex junctions can help us to understand the structural basis of specificity, and aid in the design of optimal antigene oligonucleotides. The solution structures of the junction triplexes d(GAGAGACGTA)-X-(TACGTCTCTC)-X-(CTCTCT) and d(CTCTCT)-X-(TCTCTCAGTC)-X-(GACTGAGAGA) (where X is bis(octylphosphate) and nucleotides in the triplex regions are underlined) have been solved using nuclear magnetic resonance (NMR) spectroscopy. The structure is characterised by significant changes in the conformation of the purine residues, asymmetry of the 5' and 3' junctions, and variations in groove widths associated with the positive charge of the protonated cytosine residues in the third strand. The thermodynamic stability of triplexes with either a 5' or a 3'CH+ is higher than those with a terminal thymidine. The observed sequence dependence of the triplex structure, and the distortions of the DNA at the 5' and 3' termini has implications for the design of optimal triplex-forming sequences, both in terms of the terminal bases and the importance of including positive charges in the third strand. Thus, triplex-stabilising ligands might be designed that can discriminate between TA x T-rich and CG x C+-rich sequences that depend not only on charge, but also on local groove widths. This could improve the stabilisation and specificity of antigene triplex formation.

Inhibition of PNA triplex formation by N4-benzoylated cytosine

The synthesis of N-((N4-(benzoyl)cytosine-1-yl)acetyl)- N -(2-Boc-aminoethyl)glycine (CBz) and the incorporation of this monomer into PNA oligomers are described. A single CBzresidue within a 10mer homopyrimidine PNA is capable of switching the preferred binding mode from a parallel to an antiparallel orientation when targeting a deoxyribonucleotide sequence at neutral pH. The resulting complex has a thermal stability equal to that of the corresponding PNA-DNA duplex, indicative of a strong destabilization of Hoogsteen strand PNA binding due to steric interference by the benzoyl moieties. Accordingly, incorporation of the CBz residue into linked PNAs (bis-PNAs) results in greatly reduced thermal stability of the formed PNA:DNA complexes. Thus, incorporation of the CBz monomer could eliminate the stability bias of triplex-forming sequences in PNA used in hybridization arrays and combinatorial library formats. Furthermore, it is shown that the benzoyl moiety does not severely interfere with Watson-Crick hydrogen bonding, thereby presenting an interesting route for novel cytosine modifications.

Recruitment of transcription factors to the target site by triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.

Suppression of insulin-like growth factor type I receptor by a triple-helix strategy inhibits IGF-I transcription and tumorigenic potential of rat C6 glioblastoma cells

Homopurine (AG) and homopyrimidine (CT) oligodeoxyribonucleotides predicted to form triple-helical (triplex) structures have been shown to specifically suppress gene expression when supplied to cultured cells. Here we present evidence that homopurine RNA (effector) sequences designed to form a triplex with a homopurine. homopyrimidine sequence 3' to the termination codon of the insulin-like growth factor type I receptor (IGF-IR) structural gene can efficiently suppress IGF-IR gene transcription. Transfection vectors were constructed to drive transcription of either AG or CT variant triplex-forming strands. To increase the probability of obtaining stable transfectants with adequate expression of effector sequences, these were designed to be transcribed together with cDNA sequences conferring neomycin resistance as a fusion transcript. Rat C6 glioblastoma cells transfected with the AG variant showed dramatic reduction of IGF-IR transcripts compared with untransfected cells. The AG transfectants also exhibited marked down-regulation of the IGF-I, and an enhanced accumulation of serine protease inhibitor nexin-I mRNA. Similar changes in gene expression were observed following transfection of C6 cells with constructs transcribing antisense RNA to IGF-IR transcripts, but were not observed in C6 cells transfected with either the CT triplex variant or with vector lacking triplex-forming sequences. Moreover, C6 cells transfected with AG triplex variant displayed a dramatic inhibition of tumor growth when injected into nude mice. The results suggest that a triple-helix strategy can be used to inhibit transcription elongation of the IGF-IR gene, and emphasize the efficacy of triplex-mediated gene inhibition in an animal model.

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/ homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.

Structural Optimization of Non-Nucleotide Loop Replacements for Duplex and Triplex DNAs.

Described are studies systematically exploring structural effects in he use of ethylene glycol (EG) oligomers as non-nucleotide replacements for nucleotide loops in duplex and triplex DNAs. The new structurally optimized loop replacements are more stabilizing in duplexes and triplexes than previously described EG-based linkers. A series of compounds ranging in length from tris(ethylene glycol) to octakis(ethylene glycol) are derivatized as monodimethoxytrityl ethers on one end and phosphoramidites on the other, to enable their incorporation into DNA strands by automated methods. These linker molecules span lengths ranging from 13 to 31 Å in extended conformation. They are incorporated into a series of duplex-forming and triplex-forming sequences, and the stabilities of the corresponding helixes are measured by thermal denaturation. In the duplex series, results show that the optimum linker is the one derived from heptakis(ethylene glycol), which is longer than most previous loop replacements studied. This affords a helix with greater thermal stability than one with a natural T(4) loop. In the triplex series, the loop replacements were examined in four separate situations, in which the loop lies in the 5' or 3' orientation and the central purine target strand is short or extends beyond the loop. Results show that in all cases the loop derived from octakis(ethylene glycol) (EG(8)) gives the greatest stability. In the cases where the target strand is short, the EG(8)-linked probe strands bind with affinities in some cases greater than those with a natural pentanucleotide (T(5)) loop. For the cases where the target strand extends beyond the linker, the EG(8)-linked strands are much lower in the 5' loop orientation than in the 3' loop orientation. It is found that extension by one additional nucleotide in one of the bonding domains in the EG-linked series can result in considerably greater stabilities with long target strands. Overall, the data show that optimum loop replacements are longer than would be expected from simple distance analysis. The results are discussed in relation to expected lengths and geometries for double and triple helixes. The findings will be usefull in the design of synthetically modified nucleic acids for use as diagnostic probes, as biochemical tools, and as potential therapeutic agents.

Antiparallel, intramolecular triplex DNA stimulates homologous recombination in human cells.

The DNA motif 5'-AAGGGAGAAXGGGTATAGGGYAAGAGGGAA-3' (named XY32) is an H-palindrome and has been shown to undergo a superhelix-induced, pH-dependent structural transition to H-form (pyrimidine purineo pyrimidine triplex) DNA when X = Y = A (AA32) or X = Y = G (GG32), but when X = A and Y = G (AG32) or X = G and Y = A (GA32), the transition is much more difficult [Mirkin, S. (1987) Nature (London) 330, 495-497]. Furthermore, AA32, GG32, and GA32 triplexes have the proper sequence structure to potentially form pyrimidineopurineopurine (*H-form) triplexes, but AG32 does not [Beal, P. A. & Dervan, P. B. (1992) Nucleic Acids Res. 20, 2773-2776]. Using an in vivo plasmid-plasmid recombination assay system in cultured human cells, we have found that AA32, GA32, and GG32 stimulate homologous recombination between plasmids 3- to 5-fold when both recombination substrates contain these triplex-forming sequences, whereas AG32, which differs from the others by only 1 or 2 bp, does not significantly affect the frequency of recombination. Double-strand breaks, which destroy supercoiling, nullify the stimulation. Therefore, stimulation of homologous recombination between plasmids containing these sequences correlates with their triplex-forming potential. Crosses in which the triplex-forming sequence is inserted into only one substrate exhibit an intermediate stimulation, suggesting that the inserts are acting alone as intramolecular triplexes.

Protection of DNA sequences by triplex-bridge formation.

We have demonstrated that the DNA sequence between two triplex-forming polypurine.polypyrimidine (Pu.Py) tracts was protected from DNA modifying enzymes upon formation of triplex DNA structures with an oligodeoxyribonucleotide in which two triplex-forming Pu or Py tracts were placed at the termini (triplex-bridge formation). In model experiments, when two triplex structures were formed between double-stranded DNA with the sequence (AG)17-(N)18-(T)34, and an oligodeoxyribonucleotide, (T)34-(N)18-(GA)17, not only the Pu.Py tracts but also the 18 bp non-Pu.Py sequence in the duplex DNA between the tracts was protected from restriction enzymes, HpaII methylase and DNase I. This protection occurred only when both of the Pu.Py tracts were involved as triplexes. The length of the tracts could be as short as 21 bp, while the difference in length between the non-Pu.Py sequences on the duplex and the oligodeoxyribonucleotide should be within 10 nucleotides. The efficiency of protection was enhanced in the presence of a cationic detergent, cetyltrimethylammonium bromide, during triplex formation. Protection was also observed with another type of the triplex bridge formed between (G)34 and (T)34 tracts with an oligodeoxyribonucleotide, (T)34-(N)20-(G)34. These findings suggest that the protection of specific DNA sequences from enzymes by triplex-bridge formation can be applied to any DNA sequence by placing it between two triplex-forming sequences.

Suicidal nucleotide sequences for DNA polymerization.

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.

Environmental influences on the in vivo level of intramolecular triplex DNA in Escherichia coli.

We describe an assay for detecting intramolecular triple-stranded DNA in living cells. The assay involves quantitative analysis of the differential photobinding of 4,5',8-trimethylpsoralen to 5'TA and 5'AT dinucleotides in a region of plasmid DNA containing the triplex-forming sequence (GA)7TA(GA)7. Psoralen photobinds to the central 5'TA within the (GA)7TA(GA)7 sequence in duplex DNA but not when the sequence exists as an intramolecular triplex structure. The reactivity of photobinding sites in regions flanking the intramolecular triplex-forming sequence, those that comprise the duplex-triplex junctions, either increase or decrease upon formation of the intramolecular triplex structure from duplex DNA. The pattern of trimethylpsoralen reactivity provides an indication of the conformation of the (GA)7TA(GA)7 sequence in plasmid DNA. The formation of the intramolecular triplex structure is dependent on both pH and negative superhelical density in vitro. The fraction of the (GA)7TA(GA)7 sequence that existed as an intramolecular triplex structure in vivo was dependent on the level of DNA supercoiling in vivo and changes in growth conditions which influence the intracellular pH. The Hy3 isomer was detected in living Escherichia coli cells.

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.