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The University of Southampton
Biological Sciences

Research project: DNA sequence recognition by triple helix formation

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One means of achieving precise DNA sequence recognition over several base pairs involves the formation of intermolecular DNA triple helices.

Triple-helical nucleic acids are formed by binding an oligonucleotide within the major groove of duplex DNA. These complexes offer the possibility of designing oligonucleotides which bind to duplex DNA with considerable sequence specificity. However, triple-helix formation with natural nucleotides is limited by (i) the requirement for low pH, (ii) the requirement for homopurine target sequences, and (iii) their relatively low affinity. We have prepared modified oligonucleotides to overcome these limitations, including the addition of positive charges to the sugar and/or base, the inclusion of cytosine analogues, the development of nucleosides for recognition of pyrimidine interruptions and the attachment of one or more cross-linking groups. By these means we are able to generate triplexes which have high affinities at physiological pH at sequences that contain pyrimidine interruptions.

Triple-helical nucleic acids can be generated by binding an oligonucleotide within the major groove of duplex DNA and are stabilized by the formation of hydrogen bonds between the third-strand bases and exposed groups on the purine strand of duplex DNA. The third strand can be oriented in either the parallel or anti-parallel direction, relative to the purine strand, although parallel triplexes are usually more stable, especially at low pH. In the parallel motif, GC is recognized by protonated cytosine, while AT is bound by thymine, generating C+.GC and T.AT triplets. These structures offer the possibility of designing oligonucleotides which bind to duplex DNA with considerable sequence specificity. However, triple helix formation with natural nucleotides is limited by (i) the requirement for low pH (necessary for formation of the C+.GC triplet), (ii) the requirement for homopurine target sequences, and (iii) the relatively low affinity due to charge repulsion between the polyanionic strands. We have prepared heavily modified oligonucleotides (while retaining the phosphodiester backbone) in order to overcome these limitations, which include the addition of positive charges to the sugar and/or base, the inclusion of cytosine analogues, the development of nucleosides for recognition of pyrimidine interruptions and the attachment of one or more cross-linking groups (psoralen). By these means we are able to generate triplexes, which have high affinities at physiological pH at sequences that contain pyrimidine interruptions.

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Molecular and Cellular Biosciences
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