We established a robust mRNA display platform for developing protein binding domains that efficiently and selectively associate with various analytes, such as peptides, proteins, small molecules, and complexes.
These binding domains have broad applications in research, diagnosis, and therapy. mRNA display relies on a puromycin molecule that mimics amino-acylated tRNA and forms a stable amide bond with nascent polypeptides, creating the linkage between phenotype and genotype. Compared to the other in vivo (bacteria, yeast, phage, etc) and in vitro display (ribosome, DNA display) systems, mRNA display has numerous advantages. It supports large libraries (up to 1013 variants) and allows extensive exploration of sequence space while offering flexibility in the choice of buffer conditions and operational temperatures. Further, mRNA display is significantly faster than the selection systems involving cells, such as phage and yeast display. After our optimization, a single selection round of mRNA display can be completed within hours. Typically, it involves 4 to 5 rounds of selection to eliminate non-functional sequences and enrich binding proteins.
Using this technique, we have successfully selected high-affinity and selective binders from libraries of stabilised FN3 monobody domains known as Fn3con. The selected binders can be integrated with a generic protein switch platform to create biosensors for monitoring protein biomarkers (such as liver toxicity and heart failure disease) as well as antibiotics and small molecule drugs.
