MRNA display

mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to an immobilized target in a selection step (affinity chromatography). The mRNA-protein fusions that bind well are then reverse transcribed to cDNA and their sequence amplified via a polymerase chain reaction. The result is a nucleotide sequence that encodes a peptide with high affinity for the molecule of interest.

Puromycin is an analogue of the 3’ end of a tyrosyl-tRNA with a part of its structure mimics a molecule of adenosine, and the other part mimics a molecule of tyrosine. Compared to the cleavable ester bond in a tyrosyl-tRNA, puromycin has a non-hydrolysable amide bond. As a result, puromycin interferes with translation, and causes premature release of translation products.

Figure 1. mRNA-Polypeptide Fusion Formation. a. Ribosome moves along the mRNA template and nascent peptide is being made. When the ribosome reaches the 3’ end of the template, the fused puromycin will enter the A site of the ribosome. b. The mRNA-polypeptide fusion is released.

All mRNA templates used for mRNA display technology have puromycin at their 3’ end. As translation proceeds, ribosome moves along the mRNA template, and once it reaches the 3’ end of the template, the fused puromycin will enter ribosome’s A site and be incorporated into the nascent peptide. The mRNA-polypeptide fusion is then released from the ribosome (Figure 1).

To synthesize an mRNA-polypeptide fusion, the fused puromycin is not the only modification to the mRNA template.[1] Oligonucleotides and other spacers need to be recruited along with the puromycin to provide flexibility and proper length for the puromycin to enter the A site. Ideally, the linker between the 3’ end of an mRNA and the puromycin has to be flexible and long enough to allow the puromycin to enter the A site upon translation of the last codon. This enables the efficient production of high-quality, full-length mRNA-polypeptide fusion. Rihe Liu et al. optimized the 3’-puromycin oligonucleotide spacer. They reported that dA25 in combination with a Spacer 9 (Glen Research), and dAdCdCP at the 5’ terminus worked the best for the fusion reaction. They found that linkers longer than 40 nucleotides and shorter than 16 nucleotides showed greatly reduced efficiency of fusion formation. Also, when the sequence rUrUP presented adjacent to the puromycin, fusion did not form efficiently.[2]

In addition to providing flexibility and length, the poly dA portion of the linker also allows further purification of the mRNA-polypeptide fusion due to its high affinity for dT cellulose resin.[3] The mRNA-polypeptide fusions can be selected over immobilized selection targets for several rounds with increasing stringency. After each round of selection, those library members that stay bound to the immobilized target are PCR amplified, and non-binders are washed off.

  1. ^ Roberts RW, Szostak JW (1997). "RNA-peptide fusions for the in vitro selection of peptides and proteins". PNAS. 94 (23): 12297–12302. Bibcode:1997PNAS...9412297R. doi:10.1073/pnas.94.23.12297. PMC 24913. PMID 9356443.
  2. ^ Liu R, Barrick JE, Szostak JW, Roberts RW (2000). "Optimized synthesis of RNA-protein fusions for in vitro protein selection". RNA-Ligand Interactions Part B. Methods in Enzymology. Vol. 318. pp. 268–93. doi:10.1016/S0076-6879(00)18058-9. ISBN 9780121822194. PMID 10889994.
  3. ^ Kurz M, Gu K, Lohse PA (2000). "Psoralen photo-crosslinked mRNA–puromycin conjugates: a novel template for the rapid and facile preparation of mRNA–protein fusions". Nucleic Acids Research. 28 (18): 83e–83. doi:10.1093/nar/28.18.e83. PMC 110755. PMID 10982894.