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

Some regions of nucleic acid targets are not accessible to heteroduplex for mation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The sec ondary conformation of the target may be destabilised to assist its interac tion with oligonucleotide probes. To achieve this, we modified a DNA target , which has self-complementary sequence able to form a hairpin loop, by rep lacing dC with N4-ethyldeoxycytidine (d(4Et)C), which hybridises specifical ly with natural dG to give a G:C-4Et base pair with reduced stability compa red to the natural G:C base pair. Substitution by d(4Et)C greatly reduced f ormation of the target secondary structure. The lower level of secondary st ructure allowed hybridisation with complementary probes made with natural b ases. We confirmed that hybridisation could be further enhanced by modifyin g the probes with intercalating groups which stabilise the duplex.