Nucleic Acids Research, 2001, Vol. 29, No. 19 4062-4069
© 2001 Oxford University Press
New chemically reactive dsDNAs containing single internucleotide monophosphoryldithio links: reactivity of 5'-mercapto-oligodeoxyribonucleotides
Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia
Novel modified DNA duplexes with single bridging 5'-SS-monophosphoryldithio links [-OP(=O)-O–-SS-CH2-] were synthesized by autoligation of an oligonucleotide 3'-phosphorothioate and a 5'-mercapto-oligonucleotide previously converted to a 2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent template-dependent process under the conditions used and does not require strong binding of the reactive oligomers to the complementary strand. The modified internucleotide linkage, resembling the natural phosphodiester bond in size and charge density, is stable in water, easily undergoes thiol–disulfide exchange and can be specifically cleaved by the action of reducing reagents. DNA molecules containing an internal -OP(=O)-O–-SS-CH2- bridge are stable to spontaneous exchange of disulfide-linked fragments (recombination) even in the single-stranded state and are promising reagents for autocrosslinking with cysteine-containing proteins. The chemical and supramolecular properties of oligonucleotides with 5'-sulfhydryl groups were further characterized. We have shown that under the conditions of chemical ligation the 5'-SH group of the oligonucleotide has a higher reactivity towards N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than does the OH group. This autoligation, unlike disulfide bond formation, proceeds only in the presence of template oligonucleotide, necessary to provide the activated phosphate in close proximity to the SH-, OH- or phosphate function.
* To whom correspondence should be addressed at present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Tel: +1 609 258 3935; Fax: +1 609 258 2759; Email: dolinnaya{at}hotmail.com This paper is dedicated to the memory of our teacher, Prof. Zoe A. Shabarova, a pioneer in the study of chemical reactions in dsDNAs