Abstract
We have developed chromatographic and spectrophotometric assays for determining the degree of thiolation in phosphatase-resistant 5'-monothiophosphate-capped 2-5A-antisense chimeras. Concomitantly, we have explored the reactivity of this 5'-monophosphorothioate moiety with reporter reagents such as 5-iodoacetomidofluorescein and 5,5'-dithiobis(2-nitrobenzoic acid). On the basis of these reactions, analyses for 5'-monothiophosphate-functionalized 2-5A-antisense chimeras were made possible. Kinetic experiments demonstrated that oligonucleotide backbone negative charge could retard mixed disulfide formation in the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with 5'-monothiophosphorylated 2-5A-antisense chimeras.
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