mRNA display: Diversity matters during in vitro selection

David Wilson, Anthony Keefe, and Jack Szostak have made a wonderful contribution to the changing world of peptide and protein in vitro evolution and selection. In a previous issue of PNAS (1), they demonstrated that the expected (and observed) weak affinities of peptides (from, for example, bead or phage display) can be enhanced by using “mRNA display.” The present work is a large step forward. The work builds on the earlier work of Roberts and Szostak (2). These authors, after inspecting the state of phage display for peptide evolution and selection, realized that the limiting feature of phage display was the “bacterial transformation requirement”; phage display libraries are thus limited, effectively, to <109 different peptides in the starting library. Synthetic peptide display and selection on beads is not better; screening and selection of beads with one peptide sequence per bead cannot be done easily with 109 beads. The solution to this limitation was to move toward ribosome display (3) or “mRNA display.” In either embodiment, the solution is technically complex but conceptually simple. One prepares libraries of randomized mRNAs (containing on the order of 1013 to 1014 different messages), with each randomized message being flanked by fixed sequences to allow amplification after rounds of selection. [These mRNA libraries would be perfect libraries for in vitro evolution and selection of RNA aptamers, but the goal of these experiments is peptides and proteins (encoded by the mRNA libraries), not aptamers (4)]. In the case of ribosome display, the mRNA is stalled after translation of the randomized codons to allow selection of the appropriate peptides protruding from the ribosome that had just read each mRNA, with specific mRNAs still bound to each ribosome. Ribosome display is similar to phage display, with the exception that the libraries …