Nucleic Acids Res 28(20): 3904-3909

Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

^{To whom correspondence should be addressed. Tel: +44 1865 275282; Fax: +44 1865 275283; Email: ems@bioch.ox.ac.uk Present address: Hong-Khanh Nguyen, Proteus, Parc Georges Besse, Allée Graham Bell, 30 000 Nîmes, France}

Received 2000 Jun 28; Revised 2000 Aug 21; Accepted 2000 Aug 21.

Abstract

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson–Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N4-ethyldeoxycytidine (d^{C), which hybridises specifically with natural dG to give a G:}^{C base pair with reduced stability compared to the natural G:C base pair. Substitution by d}^{C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.}

Abstract

ACKNOWLEDGEMENTS

The authors wish to thank T. Fell for construction of the apparatus used in the array synthesis. We are grateful to J.K. Elder for data analysis, software and help with the computer and to F.-X. Barre, K. Mir, V. Regnier and M. Sohail for helpful discussions. H.-K.N. was supported by a TMR Marie Curie research grant.

ACKNOWLEDGEMENTS

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