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Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting

Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting
Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting

We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide (major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quinoline moiety). This information can be used to design probes for detection of target nucleic acids by fluorescence or duplex melting. With cellular imaging in mind we show that 2′-OMe RNA probes with TO at the 5-position of uracil or the 2′-position of the ribose sugar are particularly effective, exhibiting up to 44-fold fluorescence enhancement against DNA and RNA, and high duplex stability. Excellent mismatch discrimination is achieved when the mispaired base is located adjacent to the TO-modified nucleotide rather than opposite to it. The simple design, ease of synthesis and favourable properties of these TO probes suggest applications in fluorescent imaging of DNA and RNA in a cellular context.

1477-0520
5943-5950
Klimkowski, Piotr
19202fc2-2dbd-4486-b01b-e0a6574b3586
De Ornellas, Sara
68bfd089-3364-4fbb-9a83-8b523e3dedfd
Singleton, Daniel
9be0cfc1-ca7a-4d93-810f-3fdc014d2c48
El-Sagheer, Afaf H.
05b8295a-64ad-4fdf-ad57-c34934a46c04
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Klimkowski, Piotr
19202fc2-2dbd-4486-b01b-e0a6574b3586
De Ornellas, Sara
68bfd089-3364-4fbb-9a83-8b523e3dedfd
Singleton, Daniel
9be0cfc1-ca7a-4d93-810f-3fdc014d2c48
El-Sagheer, Afaf H.
05b8295a-64ad-4fdf-ad57-c34934a46c04
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89

Klimkowski, Piotr, De Ornellas, Sara, Singleton, Daniel, El-Sagheer, Afaf H. and Brown, Tom (2019) Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting. Organic & Biomolecular Chemistry, 17 (24), 5943-5950. (doi:10.1039/c9ob00885c).

Record type: Article

Abstract

We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide (major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quinoline moiety). This information can be used to design probes for detection of target nucleic acids by fluorescence or duplex melting. With cellular imaging in mind we show that 2′-OMe RNA probes with TO at the 5-position of uracil or the 2′-position of the ribose sugar are particularly effective, exhibiting up to 44-fold fluorescence enhancement against DNA and RNA, and high duplex stability. Excellent mismatch discrimination is achieved when the mispaired base is located adjacent to the TO-modified nucleotide rather than opposite to it. The simple design, ease of synthesis and favourable properties of these TO probes suggest applications in fluorescent imaging of DNA and RNA in a cellular context.

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More information

Accepted/In Press date: 3 May 2019
e-pub ahead of print date: 3 June 2019

Identifiers

Local EPrints ID: 432337
URI: https://eprints.soton.ac.uk/id/eprint/432337
ISSN: 1477-0520
PURE UUID: 7eac169c-9477-44c9-a983-2ef732eb2f5a

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Date deposited: 11 Jul 2019 16:30
Last modified: 30 Jul 2019 16:30

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Contributors

Author: Piotr Klimkowski
Author: Sara De Ornellas
Author: Daniel Singleton
Author: Afaf H. El-Sagheer
Author: Tom Brown

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