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 (gfp(amber141) or gfp(amber178)) and wild-type (gfp(wild)) genes of GFP.
Sando
Shinsuke
S
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. ssando@sbchem.kyoto-u.ac.jp
Ogawa
Atsushi
A
Nishi
Teruyuki
T
Hayami
Masayoshi
M
Aoyama
Yasuhiro
Y
D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D001665:Binding Sites; D018388:Codon, Terminator; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D016153:Genes, Suppressor; D010454:Peptide Termination Factors / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D052157:Aptamers, Nucleotide; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli; C489006:prfB protein, E coli