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Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand

Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand
Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand
We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (Kd 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.
0006-2960
6714-6725
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f
Flashman, Emily
1ef04d5f-1a6c-4510-bbd6-05546e1642a2
Gowers, Darren
c86715a9-dc85-4561-816b-a73b92ec653e
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f
Flashman, Emily
1ef04d5f-1a6c-4510-bbd6-05546e1642a2
Gowers, Darren
c86715a9-dc85-4561-816b-a73b92ec653e

Fox, Keith R., Flashman, Emily and Gowers, Darren (2000) Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand. Biochemistry, 39 (22), 6714-6725. (doi:10.1021/bi992773+).

Record type: Article

Abstract

We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (Kd 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.

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More information

Submitted date: 3 December 1999
Published date: 1 June 2000

Identifiers

Local EPrints ID: 56069
URI: https://eprints.soton.ac.uk/id/eprint/56069
ISSN: 0006-2960
PURE UUID: 0cc4145b-6c2a-418e-85f8-c77201c93bcd
ORCID for Keith R. Fox: ORCID iD orcid.org/0000-0002-2925-7315

Catalogue record

Date deposited: 06 Aug 2008
Last modified: 14 Mar 2019 01:56

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