Directed mRNA display

X2. Xu, L., Aha, P., Gu, K., et al.. Directed evolution of high-affinity antibody mimics using mRNA display. Chem. Biol. 9, 933-942 (2002). 

In ribosome display, the physical link between genotype and phenotype is accomplished by mRNA-ribosome—protein complexes, which are directly used for selection. If a library of different mRNA molecules is translated, a protein library results in which each protein is produced from its own mRNA and remains connected to it. Since these complexes of the proteins and their encoding mRNAs are stable for several days under the appropriate conditions, very stringent selections can be performed. As all steps of ribosome display are carried out in vitro, reaction conditions of the individual steps can be tailored to the requirements of the protein species investigated, as well as the objectives of the selection or evolution experiment. Application of ribosome display has produced scFv fragments of antibodies with affinities in the picomolar range from libraries prepared from immunized mice (Hanes et al., 1998) and more recently from a naive, completely synthetic library (Hanes et al., 2000), and has been used to evolve improved off-rates and stability (Jermutus et al., 2000). 

Fig. 2. Ribosome display. A library of proteins (e.g., scFv fragments of antibodies) is transcribed and translated in vitro. The resulting mRNA lacks a stop codon, giving rise to linked mRNA-ribosome-protein complexes. These are directly used for selection on the immobilized target. The mRNA incorporated in bound complexes is eluted and purified. RT-PCR can introduce mutations and yields a DNA pool enriched for binders that can be used for the next iteration.

Protein-ligand interactions can not only be secreened or selected in vitro, but also can be directly characterized for particular interaction partners. Nemoto et al. (1999) applied the mRNA-peptide fusion technology to fluorescently label the displayed proteins in order to study protein-protein interactions by fluorescence polarization measurements. 

Ribosome display uses the unmodified DNA library, which is transcribed and translated in vitro. The link occurs between the evolved protein and mRNA, and is achieved by stalling the translating ribosome at the end of the mRNA, which lacks a stop codon. Without a stop codon, the protein is not released by the ribosome, and the complex formed by the mRNA, protein (usually correctly folded) and ribosome is used directly for selection against an immobilized target. This method is shown schematically in Figure 8.7(a). 

Figure 2 In ribosome display, mRNA (A) extracted from a cell is converted into a cDNA library (B) is transcribed back into mRNA with no stop codons. Prokaryotic or eukaryotic proteosomes are added and the ribosome then travels down the mRNA (C) translating until it reaches the end of the mRNA molecule (D), where the ribosome halts. With no stop codon, the release factor proteins cannot bind and so the protein, ribosome, and mRNA are physically associated and can be stabilized by high Mg2+ and low temperatures. This complex could then be bound directly to an immobilized natural product (E), the nonbinding library members washed away and the bound members eluted with EDTA (F), which destabilizes the ribosomal complexes by removing Mg2+. The purified sublibrary is converted into cDNA by reverse transcription (RT-PCR) and amplified by regular PCR (B). The/n vitro transcription and translation can be repeated for another round of selection or the cDNA can be analyzed by agarose electrophoresis and/or sequencing.

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