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 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 N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)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.
Nguyen
H K
HK
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Southern
E M
EM
D020029:Base Pairing / Q000187:drug effects; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003853:Deoxyribonucleosides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D007364:Intercalating Agents / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000187:drug effects / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D013696:Temperature; D013816:Thermodynamics
D015342:DNA Probes; D003853:Deoxyribonucleosides; D007364:Intercalating Agents; D004247:DNA