We have developed chromatographic and spectrophotometric assays for de
termining the degree of thiolation in phosphatase-resistant 5'-monothi
ophosphate-capped 2-5A-antisense chimeras. Concomitantly, we have expl
ored the reactivity of this 5'-monophosphorothioate moiety with report
er reagents such as 5-iodoacetomidofluorescein and 5,5'-dithiobis(2-ni
trobenzoic acid). On the basis of these reactions, analyses for 5'-mon
othiophosphate-functionalized 2-5A-antisense chimeras were made possib
le. Kinetic experiments demonstrated that oligonucleotide backbone neg
ative charge could retard mixed disulfide formation in the reaction of
5,5'-dithiobis(2-nitrobenzoic acid) with 5'-monothiophosphorylated 2-
5A-antisense chimeras.