Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation.
- 1 March 1988
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 85 (5), 1349-1353
- https://doi.org/10.1073/pnas.85.5.1349
Abstract
A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural (.beta.) anomer of thymidine or the synthetic (.alpha.) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA .cntdot. dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence as observed generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocross-linked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both .alpha. and .beta. oligothymidylates can recognize a DNA double helix containing an oligo(dA) .cntdot. oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) .alpha. oligothymidylates form helices with a complementary single-stranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, .beta. oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that is possible to recognize an oligopurine .cntdot. oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photchemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.This publication has 33 references indexed in Scilit:
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