Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies

Taishi Kondo; Minori Eguchi; Nariaki Tsuzuki; Naoya Murata; Tomoshige Fujino; Gosuke Hayashi; Hiroshi Murakami

doi:10.21769/BioProtoc.4125

Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies

Bio Protoc. 2021 Aug 20;11(16):e4125. doi: 10.21769/BioProtoc.4125.

Authors

Taishi Kondo¹, Minori Eguchi¹, Nariaki Tsuzuki¹, Naoya Murata¹, Tomoshige Fujino¹, Gosuke Hayashi^{1

2}, Hiroshi Murakami^{1

3}

Affiliations

¹ Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan.
² Japan Science and Technology Agency (JST), PRESTO, Japan.
³ Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Nagoya, Japan.

Abstract

Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody mRNA libraries with greater than 10¹³ theoretical diversity. First, we synthesized two long single-stranded DNAs that corresponded to fragments of monobody DNA, with random codons in the BC and FG loops. These oligonucleotides were ligated by T4 DNA ligase with the support of guide oligonucleotides containing 3' ends that were protected by a modification. After amplifying the product DNAs by PCR, one end of each DNA fragment was digested with the type II restriction enzyme BsaI, and the resulting DNA fragments were ligated using T4 DNA ligase. After amplification of the DNA product, mRNAs were synthesized by T7 RNA polymerase. This method is simple and could be used for the preparation of mRNA libraries for various antibody-like proteins. Graphic abstract: Construction of a highly diverse mRNA library.

Keywords: Antibody-like protein; In vitro selection; Monobody; TRAP display; mRNA library.