AN IN-VITRO MESSENGER-RNA BINDING ASSAY AS A TOOL FOR IDENTIFYING HYBRIDIZATION-COMPETENT ANTISENSE OLIGONUCLEOTIDES

The apparent dissociation constants for 32 phosphodiester and 5 phosph orothioate antisense oligodeoxyribonucleotides (ODN) targeted to murin e tumor necrosis factor-alpha (mTNF-alpha) mRNA were determined using a gel-shift binding assay, In this assay, radiolabeled ODN were hybrid ized in solution to a structured mRNA transcript generated in vitro. F ree ODN was resolved from bound ODN on a two-phase discontinuous polya crylamide gel. Excision of gel slices containing free ODN or bound ODN , followed by Cerenkov counting of the slices, was used to prepare app arent binding isotherms for each ODN. Apparent dissociation constants for the anti-mTNF-alpha ODN varied from >100 mu M to 0.4 nM. Slight di fferences in RNA target site position resulted in significant differen ces in apparent affinity, particularly for shorter (12-mer) ODN. This binding assay provides an empirical means for selecting ODN sequences possessing high affinity for a target RNA and lends itself to a high t hroughput assay in which all possible antisense sequences of a given l ength can be evaluated to obtain the better binders for use in cell cu lture or in vivo.