mRNA display

The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage.

The mRNA-protein fusions that bind well are then reverse transcribed to cDNA and their sequence amplified via a polymerase chain reaction.

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.

[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.

After each round of selection, those library members that stay bound to the immobilized target are PCR amplified, and non-binders are washed off.

A DNA library for any protein or small peptide of interest can be synthesized by solid-phase synthesis followed by PCR amplification.

The ligated mRNA-DNA-puromycin library is translated in Red Nova Lysate (Novagen) or E. coli S30 Extract System (Promega), resulting in polypeptides covalently linked in cis to the encoding mRNA.

Following binding selection, those library members that stay bound to the immobilized target are PCR amplified.

The PCR amplification step will enrich the population from the mRNA-display library that has higher affinity for the immobilized target.

In contrast, the puromycin DNA spacer linker used in mRNA display technology is much smaller comparing to a ribosome.

[6] Nine years later, Fukuda and colleagues chose mRNA display method for in vitro evolution of single-chain Fv (scFv) antibody fragments.

And they concluded that mRNA display has the potential for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

Roberts and coworkers have demonstrated that unnatural peptide oligomers consisting of an N-substituted amino acid can be synthesized as mRNA-polypeptide fusions.

[13] N-substituted amino acid-containing peptides have been associated with good proteolytic stability and improved pharmacokinetic properties.

This work indicates that mRNA display technology has the potential for selecting drug-like peptides for therapeutic usage resistant to proteolysis.

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.
Figure 2. Splint Aid Single Stranded mRNA/DNA T4 DNA Ligase Ligation
Figure 3. Selection Cycle.