One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA

ACS Comb Sci. 2011 Sep 12;13(5):478-85. doi: 10.1021/co2000295. Epub 2011 Jul 28.

Abstract

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System
  • DNA, Complementary / chemical synthesis*
  • DNA, Complementary / chemistry
  • Magnetic Phenomena
  • Peptide Library
  • Puromycin / chemistry*
  • RNA, Messenger / chemical synthesis*
  • RNA, Messenger / chemistry

Substances

  • DNA, Complementary
  • Peptide Library
  • RNA, Messenger
  • Puromycin