In vitro selection of RNA aptamer agianst Escherichia coli release factor 1

A pool of 84-nt RNAs containing a randomized sequence of 50 nt was selected against gel-immobilized Escherichia coli release factor 1 (RF-1) responsible for translation termination at amber (UAG) stop codon. The strongest aptamer (class II-I) obtained from 43 clones bound to RF-1, but not to UAA/UGA-targeting RF-2, with K-d = 30 +/- 6 nM (SPR). A couple of impaired hairpin domains in the aptamer were suggested as the sites of attachment of RF-1. By binding to and hence inhibiting the action of RF-1 specifically or bio-orthogonally, aptamer class II-I enhanced the amber suppression efficiency in the presence of an anticodon-adjusted (CUA) suppressor tRNA without practically damaging the protein translation machinery of the cell-free extract of E coli, as confirmed by the translation of amber-mutated (gfp(amber141) or gfp(amber178)) and wild-type (gfp(wild)) genes of GFP. (c) 2006 Elsevier Ltd. All rights reserved.