cDNA Display: A Stable and Simple Genotype–Phenotype Coupling Using a Cell-Free Translation System

  • Hidenao Arai
  • Shigefumi Kumachi
  • Naoto NemotoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)


A cDNA display method was developed based on the mRNA display method to increase its stability and efficiency for the directed evolution of various kinds of peptides and proteins. In this method, the puromycin-linker is a key molecule to realize smart genotype–phenotype coupling. A recently improved puromycin-linker and its use were explained in detail for the in vitro selection of peptides and proteins using the cDNA display method.

Key words

cDNA display mRNA display In vitro selection Directed evolution Puromycin-linker cnvK Photocrosslinking 


  1. 1.
    Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H (1997) In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett 414:405–408CrossRefGoogle Scholar
  2. 2.
    Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94:12297–12302CrossRefGoogle Scholar
  3. 3.
    Nishigaki K, Taguchi K, Kinoshita Y, Aita T, Husimi Y (1998) Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase. Mol Divers 4:187–190CrossRefGoogle Scholar
  4. 4.
    Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N (2009) cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Nucleic Acids Res 37:e108CrossRefGoogle Scholar
  5. 5.
    Mochizuki Y, Biyani M, Tsuji-Ueno S, Suzuki M, Nishigaki K, Husimi Y, Nemoto N (2011) One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA. ACS Comb Sci 13:478–485CrossRefGoogle Scholar
  6. 6.
    Mochizuki Y, Suzuki T, Fujimoto K, Nemoto N (2015) A versatile puromycin-linker using cnvK for high-throughput in vitro selection by cDNA display. J Biotechnol 212:174–180CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Hidenao Arai
    • 1
  • Shigefumi Kumachi
    • 1
  • Naoto Nemoto
    • 1
    • 2
    Email author
  1. 1.Epsilon Molecular Engineering, Inc.SaitamaJapan
  2. 2.Graduate School of Science and EngineeringSaitama UniversitySaitamaJapan

Personalised recommendations