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Nucleic Acids Research, 2000, Vol. 28, No. 20 3904-3909
© 2000 Oxford University Press

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

Hong-Khanh Nguyen and Edwin M. Southern*

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

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 (d4EtC), which hybridises specifically with natural dG to give a G:4EtC base pair with reduced stability compared to the natural G:C base pair. Substitution by d4EtC 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.

* 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


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