GoKawiiltRNA in vitro production
Oligonucleotides containing tRNA under a T7 promoter and followed by an HDV ribozyme61 were synthesized by IDT as high-fidelity DNA microchip oligo pools from 254–275 nucleotides in length, and dsDNA was amplified in a single PCR reaction for each pool. These tRNA pools included 48 unique E. coli isoacceptor tRNAs with all combinations of base 75, all combinations of bases 74 and 75 (degeneracy introduced with machine mixing), and all M. alvus and M. mazei isoacceptor tRNAs. IVT reactions were carried out at 37 °C using the amplified tRNA dsDNA as template using NEB HiScribe T7 High Yield RNA Synthesis Kit, with addition of ATP, CTP, GTP, UTP, reaction buffer, 200 ng dsDNA template and T7 RNA polymerase. After reverse transcription, polynucleotide kinase treatment was performed to dephosphorylate the tRNA 3′ terminus62. tRNA produced by IVT was purified and size-selected using SPRIselect beads. The tRNA was refolded by heating to 80 °C for 5 min and slow cooling to room temperature. At 50 °C, MgCl 2 was added to a final concentration of 10 mM.
For amplification of individual tRNAs from a tRNA pool, a unique primer was designed for each tRNA covering the T7 promoter and 5′ unique sequence of the tRNA. The same reverse primer was used for all tRNAs as for the pooled amplification. Alternatively, individual tRNA sequences were synthesized and amplified.
tRNA aminoacylation in cell-free lysates
A total of 40–120 µg of IVT tRNA libraries were incubated in 200 μl NEBExpress Cell-free E. coli Protein Synthesis System (New England Biolabs), or a custom cell lysate translation system we prepared (see below), and incubated for 2 h at 37 °C. Alternatively, 40–120 µg of IVT tRNA libraries were incubated in the NEB PURExpress Δ(aa, tRNA) kit and incubated for 2 h at 37 °C.
tRNA extraction
Cell pellets were suspended in 1 ml TRIzol (Thermo Fisher) and frozen at −80 °C. 1-Bromo-3-chloropropane (1/10 volume) was added, and the samples were vortexed and centrifuged at 15,000 × g for 15 min at 4 °C. The aqueous phase was transferred to a new tube containing 400 μl of 70% ethanol. Short RNAs were then isolated using a modified RNeasy MinElute Cleanup Kit protocol (Qiagen) to size-select for RNAs <200 nt in length. Samples were centrifuged through MinElute spin column at 12,000 × g for 5 min at room temperature. The flow-through was added to 450 μl of 100% ethanol and centrifuged in a new MinElute spin column at 12,000 × g for 1 min at room temperature. The column was washed three times with 80% ethanol in 50 mM sodium acetate and dried with open caps at 12,000 × g for 5 min. Samples were eluted in 50 mM sodium acetate and 1 mM EDTA.
Periodate oxidation and β-elimination
Periodate oxidation was carried out with 10 μg total RNA isolated above in 10 mM sodium acetate and 50 mM NaIO 4 . The reaction was incubated at 22 °C for 30 min and quenched with 100 mM d-glucose for 5 min. The samples were size-selected using SPRIselect beads (Beckman Coulter) and eluted in RNase-free water. β-Elimination and deacylation were carried out in 60 mM sodium tetraborate at 45 °C for 90 min. RNA was purified and size-selected using SPRIselect beads. The 3′ phosphoryl group was removed using New England Biolabs T4 Polynucleotide Kinase kit following the protocol of the manufacturer. Dephosphorylated RNA was purified using SPRIselect beads.
tRNA library preparation and sequencing
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