Streamlined Protocol for mRNA Display

Abstract

mRNA display is a powerful method for in vitro directed evolution of polypeptides, but its time-consuming, technically demanding nature has hindered its widespread use. We present a streamlined protocol in which lengthy mRNA purification steps are replaced with faster precipitation and ultrafiltration alternatives; additionally, other purification steps are entirely eliminated by using a reconstituted translation system and by performing reverse transcription after selection, which also protects input polypeptides from thermal denaturation. We tested this procedure by performing affinity selection against Her2 using binary libraries containing a non-specific designed ankyrin repeat protein (DARPin) doped with a Her2-binding DARPin (dopant fraction ranging from 1:10 to 1:10,000). The Her2-binding DARPin was recovered in all cases, with an enrichment factor of up to two orders of magnitude per selection round. The time required for one round is reduced from 4–7 days to 2 days with our protocol, thus simplifying and accelerating mRNA display experiments.

Revealing protein-protein interactions at the transcriptome scale by sequencing

“…each protein. Similar to mRNA display (Roberts and Szostak, 1997;Barendt et al, 2013), SMART-display is designed to create mRNA-protein fusions, specifically by adding an amino acid analog puromycin (''P'' in the purple circle, Figure S1A) near the 3 0 end of the mRNA. The translated protein from this mRNA is then covalently linked with its mRNA when puromycin enters the A site of the ribosome and is joined to the amino acid chain.…”

“…Overview of SMART-display Because mRNA-display has been thoroughly tested (Barendt et al, 2013;Seelig, 2011), our goal is to simplify the mRNA-display process, so that mRNA-display can be performed at genome scale. Our simplification was achieved by replacing the most timeconsuming experimental step in mRNA-display (Cotten et al, 2011).…”

Conceptual and methodological advances in cell-free directed evolution

“…For example, the PURE system [3,39] is a fully defined IVTT system with a high concentration of ribosomes and low levels of detrimental nucleases and proteases, thus yielding more complex libraries of display particles. Additionally, streamlined protocols are being developed to simplify cell-free evolution [40*,41]. …”

Evolving a Peptide: Library Platforms and Diversification Strategies

“…Another technique, mRNA display, is similar to ribosome display. The essence of this technology is the employment of puromycin, an antimicrobial product that inhibits translation by mimicking the 3'-end of an aminoacyl-tRNA, conjugated to the 3' terminus of mRNA [155]. When the ribosome reaches the 3' end of the template, the newly synthesized peptide is transferred onto the puromycin, achieving genotype-phenotype linkage with its encoding mRNA.…”