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5-(1-propargylamino)-2'-deoxyuridine (U(P)): A novel thymidine analogue for generating DNA triplexes with increased stability

5-(1-propargylamino)-2'-deoxyuridine (U(P)): A novel thymidine analogue for generating DNA triplexes with increased stability
5-(1-propargylamino)-2'-deoxyuridine (U(P)): A novel thymidine analogue for generating DNA triplexes with increased stability

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (U(P)). The intramolecular triplex A6-L-T6-L-(U(P))5T (L = two octanediol residues) shows a single UV-melting transition which is > 20°higher than that of the parent triplex A6-L-T6-L-T6 at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by U(P) in a pH dependent fashion. Triplex stability increases by ~ 12 K for each T to U(P) substitution. Quantitative footprinting studies have examined the interaction of three U(P)-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the U(P)-containing oligonucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (U(P))6CU(P)T binds ~ 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.

0305-1048
1802-1809
Bijapur, Jeevan
3c723216-f0d4-45b0-bfa5-81539bee1719
Keppler, Melanie D.
6452c8eb-268a-47f4-955d-e1819df05333
Bergqvist, Simon
18c7df64-c0f3-4286-9c15-758600765916
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f
Bijapur, Jeevan
3c723216-f0d4-45b0-bfa5-81539bee1719
Keppler, Melanie D.
6452c8eb-268a-47f4-955d-e1819df05333
Bergqvist, Simon
18c7df64-c0f3-4286-9c15-758600765916
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f

Bijapur, Jeevan, Keppler, Melanie D., Bergqvist, Simon, Brown, Tom and Fox, Keith R. (1999) 5-(1-propargylamino)-2'-deoxyuridine (U(P)): A novel thymidine analogue for generating DNA triplexes with increased stability. Nucleic Acids Research, 27 (8), 1802-1809. (doi:10.1093/nar/27.8.1802).

Record type: Article

Abstract

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (U(P)). The intramolecular triplex A6-L-T6-L-(U(P))5T (L = two octanediol residues) shows a single UV-melting transition which is > 20°higher than that of the parent triplex A6-L-T6-L-T6 at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by U(P) in a pH dependent fashion. Triplex stability increases by ~ 12 K for each T to U(P) substitution. Quantitative footprinting studies have examined the interaction of three U(P)-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the U(P)-containing oligonucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (U(P))6CU(P)T binds ~ 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.

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More information

Published date: 15 April 1999
Additional Information: Funding Information: This work was supported by grants from the Cancer Research Campaign.

Identifiers

Local EPrints ID: 476352
URI: http://eprints.soton.ac.uk/id/eprint/476352
ISSN: 0305-1048
PURE UUID: dead4531-6968-495e-97b7-b67c8b36fbb3
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: 10 Aug 2023 01:31

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Contributors

Author: Jeevan Bijapur
Author: Melanie D. Keppler
Author: Simon Bergqvist
Author: Tom Brown
Author: Keith R. Fox ORCID iD

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