mRNA display: diversity matters during in vitro selection.

D 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 can be larger because amplification requires enzymology rather than bacterial transformation (specifically in the first round of phage display). In both cases the ‘‘displayed’’ peptide (or protein) is a rather small object dangling from a very large phage coat or from a very large ribosome. I have always thought that selection of peptides as binding partners (for target proteins, as is common) would be complicated by the size and surface of the displaying object (phage or ribosome); some high affinity peptides might be lost through unpredictable interactions between the displayed peptide and the enormous protein complex to which it was attached. After all, a ribosome has a mass of more than 2,000,000 Da, and typical peptide or protein libraries contain selectable molecules with masses of less than 10,000 Da. mRNA display solves the ‘‘large display object problem’’ in an elegant fashion, while maintaining the major advantage of ribosome display, which is large diversity not constrained by bacterial transformation. After translation of random peptides, as above, the encoding mRNAs are covalently pinned to the newly synthesized peptides through the puromycin reaction (the puromycin having been previously placed on the 39 end of each mRNA). Purification of the mRNA-peptide fusions, free of ribosomes, allows selection of appropriate peptides with only the encoding mRNA immediately adjacent to the peptide. In mRNA display, the mRNAs are more than ten times the size of the displayed peptide (three nucleotides per codon, plus extra nucleotides for amplification and the puromycin reaction—but this appendage is smaller than a phage or ribosome. In Fig. 1 are displayed the systems (and the sizes of those systems) that allow selection of peptides or proteins (and aptamers—see below). The authors double the size of the mRNA by doing reverse transcription before selection, for reasons discussed below. Peptide libraries about 88 aa in length were used in this publication. Gratifyingly, some of the peptides selected as binding partners to the target protein streptavidin had low dissociation constants. A few had KDs of between 5 and 10 nM, substantially lower than the KDs of peptides also aimed at streptavidin found through phage display. The authors conclude that the low KDs flow largely from the larger starting library diversity (‘‘. 104-fold more sequences sampled’’), the length of the peptides sampled (adding another 50-fold to the sample size for a 38-mer, corresponding to the different 38-mers within a peptide of 88 aa), and the slight bias in favor of HPQ sequences in their libraries (given that HPQ was a known consensus motif for streptavidin binding from previous work). I would add to their list the avoidance of presumptive interferences by phages or ribosomes (or beads, in other work) as another advantage of mRNA display. The appendage in this paper is a double-stranded and monotonous helical polyanion, rather than a textured protein-rich phage or ribosome; I think the double helix is a step in the right direction. The authors conclude that ‘‘it should be possible to increase the affinities of peptides for their targets by constraining them as loops in known protein scaffolds’’; in fact, the entire technology platform of Phylos (see their web site for more details, www.phylos.com) is aimed at exactly that approach. Interestingly, in the present work, all of the selected and sequenced clones shifted the frame (through deletion of two nucleotides or addition of one nucleotide), suggesting among other things that 1013–1014 sequences provide