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Specificity of antiparallel DNA triple helix formation

Specificity of antiparallel DNA triple helix formation
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 thought 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 hail 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.

0006-2960
15038-15048
Chandler, Simon P.
839d7f59-0eb0-4fc5-accf-6ff2bc08b243
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f
Chandler, Simon P.
839d7f59-0eb0-4fc5-accf-6ff2bc08b243
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f

Chandler, Simon P. and Fox, Keith R. (1996) Specificity of antiparallel DNA triple helix formation. Biochemistry, 35 (47), 15038-15048. (doi:10.1021/bi9609679).

Record type: Article

Abstract

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 thought 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 hail 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.

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Published date: 26 November 1996

Identifiers

Local EPrints ID: 476343
URI: http://eprints.soton.ac.uk/id/eprint/476343
ISSN: 0006-2960
PURE UUID: 87de5ecd-4dfc-4e68-81a5-17972c49227a
ORCID for Keith R. Fox: ORCID iD orcid.org/0000-0002-2925-7315

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Date deposited: 19 Apr 2023 16:46
Last modified: 17 Mar 2024 02:34

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Contributors

Author: Simon P. Chandler
Author: Keith R. Fox ORCID iD

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