MRNA display

Carey I, Noti JD (1999) Isolation of protein kinase C-alpha-reguIated cDNAs associated with breast tumor aggressiveness by differentia mRNA display. Int J Oncol 14 951-956... 

Hughes JR, Bullock SL, Ish-Horowicz D (2004) Inscuteable mRNA localization is dynein-dependent and regulates apicobasal polarity and spindle length in Drosophila neuroblasts. Curr Biol 14 1950-1956 Ja WW, Roberts RW (2004) In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display. Biochemistry 43 9265-9275... 

Wilson, D. S., Keefe, A. D., and Szostak, J. W. (2001) The use of mRNA display to select high-affinity protein-binding peptides. Proc. Natl. Acad. Sci. USA 98, 3750-3755. 

Hammond, P.W., Alpin, J. Rise, C.E., Wright, M. and Kreider, B.L. (2001) In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries. J. Biol. Chem. 276, 20898-20906. 

Lipovsek, D. and Pluckthun, A. 200A) In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51-67. 

Also called mRNA display, this method was derived from ribosome display technology. Puromycin-tagged mRNA and several additional steps must be used to achieve covalent coupling between the protein product and its mRNA, as shown in Figure 8.7(h). Puromycin, which mimics aminoacyl-tRNA (tRNA = transfer RNA), can be attached covalently to mRNA by the ribosomal machinery. 

Unique disadvantages to ribosome display relate to the inherent instability of RNA. This is a disadvantage shared by mRNA display but an advantage of ribosome display over mRNA display is that the in vitro transcription and translation can be combined into one step. This renders the mRNA less vulnerable to degradation. 

The key feature of mRNA display is that the gene (mRNA) and the encoded protein are covalently attached to each other by a puromycin-DNA linker (Figure 4). This is a key advantage over ribosome display, where the link between the mRNA and the protein is noncovalent and mediated by the ribosome. Thus mRNA display allows more stringent selection criteria to be employed that would result in the dissociation of ribosome and mRNA. 

More recently, Yanagawa and coworkers has further improved the mRNA display method by incorporation of a photocleavable 2-nitrobenzyl linker between genotype (mRNA) and phenotype (protein) to facilitate isolation of the mRNA for amplification or analysis.295 They demonstrated the utility of the photocleavable linker by also isolating FKBPla from an mRNA library using biotinylated FK506. 

These two examples, using FK506-FKBP, clearly demonstrate the potential of mRNA display for the isolation of natural product receptors and, like phage display, could be used to determine the target proteins for natural products with no known target or even no known biological activity. 

More recently, a covalently linked system exploits the replication initiator of bacteriophage P2, which covalently attaches the P2A protein to its own DNA phosphate backbone.306 This method is droplet independent, theoretically allowing libraries as large as those used in mRNA display but at present the system suffers from low complex formation efficiency ( 3%) that needs to be improved. 

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