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 phosphorothioate antisense oligodeoxyribonucleotides (ODN) targeted to murine tumor necrosis factor-alpha (mTNF-alpha) mRNA were determined using a gel-shift binding assay. In this assay, radiolabeled ODN were hybridized in solution to a structured mRNA transcript generated in vitro. Free ODN was resolved from bound ODN on a two-phase discontinuous polyacrylamide gel. Excision of gel slices containing free ODN or bound ODN, followed by Cerenkov counting of the slices, was used to prepare apparent binding isotherms for each ODN. Apparent dissociation constants for the anti-mTNF-alpha ODN varied from > 100 microM to 0.4 nM. Slight differences in RNA target site position resulted in significant differences 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 throughput assay in which all possible antisense sequences of a given length can be evaluated to obtain the better binders for use in cell culture or in vivo.