Why tRNA
Transfer RNAs (tRNAs) are key to the ribosomal readout of the genetic code. tRNA shape, stability, amino-acylation, and decoding in translation, all depend on complex post-transcriptional modifications.
High quality tRNA are crucial to all in vitro translation systems, including whole cell lysate and PURE system.
Here we share toolbox of our tRNA Foundry: methods to purify whole tRNA pools from various organisms, and methods to purify single tRNA species.
Our protocols yield tRNA with full complement of post-transcriptional modifications.
tRNA pools - total tRNA
For a long time, our community relied on MRE600 tRNA sold by Roche. Since this product was discontinued, there is an urgent need for a source of a complete tRNA pool for cell-free translation.
What and why:
Ribosomal protein synthesis is driven by tRNAs, the adaptor molecules which bridge the RNA and protein worlds. Cell-free translation systems like PURE use purified tRNAs as a reagent to complete their reactions. Historically, these tRNAs were available through commercial providers that have since disappeared. We have explored literature methods and potential strains to address this critical need.
The steps:
Growth - We simply grow organisms of interest to the mid-log phase usually associated with rapidly growing cells. Generally, this means growing to an OD600 of ~2 in an enriched medium.
Harvest - Cells are recovered through centrifugation, washed, and flash frozen before storage.
Extraction - Small RNAs are extracted from the cell using acidic phenol which partitions nucleic acids into the aqueous phase and while proteins and lipids are kept in the organic phase.
Isolation - tRNAs are separated from rRNA, mRNA, and genomic DNA through the sequential precipitation of each nucleic acid type. This is done by exploiting the different solubilities of each nucleic acid in varying concentrations of isopropanol and salts.
The Result:
From this method, we get 5-20 mg of pure tRNA that can be used directly in cell-free translation reactions. We have so far extracted tRNAs from:
- E. coli A19 (a K-12 derivative)
- E. coli BL21 (DE3)
- E. coli BL21 (DE3) Rosetta2
- Vibrio natriegens
- Mycoplasma capricolum capri
We find the greatest activity from tRNAs isolated from E. coli A19 in PURE, but we are looking to expand our portfolio of tRNA sources to potentially further improve PURE yields.
Reference:
An Expanded Repertoire of tRNA Sources for Cell-Free Protein Synthesis;
Evan M. Kalb, Russel M. Vincent, Aaron E. Engelhart, George M. Church, Katarzyna Adamala
preprint
doi.org/10.1101/2025.08.20.671396
Pick and choose: single tRNA species
Many applications can benefit from the ability to tailor custom tRNA supply, including genetic code expansion, codon bias studies and other efforts to engineer modified translation systems.
What and why:
Cell-free translation systems rely on tRNAs to mediate ribosomal protein translation. Normally, these tRNAs are supplied in bulk, containing every tRNA found in the cytoplasm across a wide range of individual abundances. Constructing custom tRNA pools with defined abundances of individual tRNAs could enable control of translation rate, improve yield, and even reassign the universal genetic code. Most efforts build custom tRNA pools from the bottom-up by synthesizing each target tRNA using in vitro transcription. This strategy is convenient and high yielding, but generates tRNAs lacking their crucially important post-transcriptional modifications. We have instead developed an approach where individual tRNAs are harvested and purified from in vivo sources to preserve their modifications. These tRNAs can then be reassembled into a custom tRNA pool.
The steps:
Express - We overexpress a tRNA of interest in from a low-copy number plasmid, enriching the native tRNA pool with a greater fraction of the target tRNA. In some tRNAs, this can increase their relative abundances from ~1% to >30% of the total pool.
Isolate - We utilize previously described tRNA isolation methods to provide the enriched tRNA pool.
Purify - From these enriched pools we purify the target tRNAs using column-based, oligonucleotide hybridization. We prepare a column coupled with an oligonucleotide specific to the tRNA of interest. The enriched tRNA pool is bound to the functionalized column using recircularized binding, where the solution is bound by cooling from a denaturing temperature (~75C) to a binding temperature (~60C). After recircularized binding, unbound, off-target tRNAs are removed from the column through washing at room temperature. Finally, the target tRNA is eluted from the column using heat elution.
Precipitate - After elution, the tRNA is precipitated and stored until its use in a custom tRNA pool.
The results:
We regularly purify 5 -10 nmoles of native single isoacceptor tRNAs.
The purified tRNAs contain their full complement of literature-described post-transcriptional modifications.
When assembled into translation reaction, the tRNAs are able to complete cell-free translation.
We have extended purification methods to tRNA important for non-canonical amino acid incorporation and to engineered tRNAs.
Reference:
Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems
Evan M. Kalb, Jose L. Alejo, Leticia Dias-Fields, Isaac Knudson, Joshua A. Davisson, Efren Maldonado, Kanokporn Chattrakun, Shangsi Lin, Alanna Schepartz, Shenglong Zhang, Scott Blanchard, Aaron E. Engelhart, Katarzyna P. Adamala
preprint
doi.org/10.1101/2025.06.10.658963
The team
The tRNA project is work in progresss in the Adamala lab, many people are engineering and improving on existing protocols and bringing in new ideas.
The project is led by Evan Kalb, Joshua Davisson and Elisabeth Edgerton.
References
tRNA pools:
An Expanded Repertoire of tRNA Sources for Cell-Free Protein Synthesis;
Evan M. Kalb, Russel M. Vincent, Aaron E. Engelhart, George M. Church, Katarzyna Adamala
preprint
doi.org/10.1101/2025.08.20.671396
Single tRNAs:
Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems;
Evan M. Kalb, Jose L. Alejo, Leticia Dias-Fields, Isaac Knudson, Joshua A. Davisson, Efren Maldonado, Kanokporn Chattrakun, Shangsi Lin, Alanna Schepartz, Shenglong Zhang, Scott Blanchard, Aaron E. Engelhart, Katarzyna P. Adamala
preprint
doi.org/10.1101/2025.06.10.658963