A Concept for Selection of Codon-Suppressor tRNAs Based on Read-Through Ribosome Display in an In Vitro Compartmentalized Cell-Free Translation System

Abstract

Here is presented a concept for in vitro selection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRNA(Ser). Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRNA(Ser).

Figure 1

(a) Schematic sequence of the tRNA-fused template with two T7 promoters. The triple N in red represents the anticodon- or anticodon-loop-randomized region. (b) Agarose gel electrophoretic assay of the template DNAs recovered from tRNA_CUA-fused template (lane 1), nonfused template lacking the tRNA domain (lane 2), a 1 : 1 mixture thereof (lanes 4, 5), or tRNA_CGA-fused template (lane 3), after coupled transcription/translation under normal (noncompartmentalized) (lanes 1–4) or compartmentalized (lane 5) conditions.

Figure 2

Rt-RD/IVC-based selection/amplification cycle. See the text for explanation.

Figure 3

(a) Summary of selection/amplification of anticodon-randomized or anticodon-matched/anticodon-loop-randomized tRNAs for Ser. The numbers of clones that possess the sequence shown are indicated in parentheses. (b) Western blotting analysis of the translation of nonfused (tRNA-lacking) mRNA templates under normal (noncompartmentalized) conditions with (lanes 9–15) or without (lane 8) tRNA; tRNA^SerU _CUA (lane9), tRNA^SerU _CGA (natural tRNA for Ser, lane 10), anticodon-loop-randomized tRNA^SerU after 0, 3, or 5 rounds of selection/amplification (lanes 11, 12, or 13, respectively), or anticodon-matched but singly loop-mutated tRNA^SerU (lanes 14 and 15). Lane 7 represents a control translation using amber-free mRNA with no use of tRNA as a positive control and lanes 1–7 are a calibration set.