In vitro selection of RNA aptamer against 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-1) obtained from 43 clones bound to RF-1, but not to UAA/UGA-targeting RF-2, with Kd = 30 ± 6 nM (SPR). A couple of unpaired 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-1 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 (gfpamber141 or gfpamber178) and wild-type (gfpwild) genes of GFP.

We successfully selected RNA aptamers against Escherichia coli release factor 1, allowing an enhanced nonsense suppression at the amber (UAG) stop codon.Figure optionsDownload as PowerPoint slide