title: One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.
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.
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Sakura-ku, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Mochizuki
authors.forename: Manish
authors.initials: M
authors.lastname: Biyani
authors.forename: Sachika
authors.initials: S
authors.lastname: Tsuji-Ueno
authors.forename: Miho
authors.initials: M
authors.lastname: Suzuki
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
mesh_terms: D002474:Cell-Free System; D018076:DNA, Complementary / Q000138:chemical synthesis* / Q000737:chemistry; D060328:Magnetic Phenomena; D019151:Peptide Library; D011691:Puromycin / Q000737:chemistry*; D012333:RNA, Messenger / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D019151:Peptide Library; D012333:RNA, Messenger; D011691:Puromycin