Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids.
A rapid selection procedure to separate low amounts of aminoacylated tRNAs from large pools of inactive variants is described. The procedure involves a three-step protocol. After initial aminoacylation of a tRNA pool, N-hydroxysuccinimide ester chemistry is applied to biotinylate the alpha-NH2 group of the amino acid bound to the 3'-end of a tRNA. The biotin tag is used to capture the derivatized tRNAs on streptavidin-conjugated magnetic beads. Variants bound to the solid phase can be amplified by RT-PCR and transcription, providing tRNAs for subsequent selection rounds.
Pütz
J
J
Institüt für Molekulare Biotechnologie, Beutenbergstrasse 11, D-07745, Jena, Germany.
Wientges
J
J
Sissler
M
M
Giegé
R
R
Florentz
C
C
Schwienhorst
A
A
D000596:Amino Acids / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000302:isolation & purification*
D000596:Amino Acids; D012346:RNA, Transfer, Amino Acyl; D001710:Biotin