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

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.

[1]  M. Machida,et al.  Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling , 2011, Molecular Brain.

[2]  Emmanuel Dias-Neto,et al.  Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis , 2009, PloS one.

[3]  M. Machida,et al.  cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA–protein fusions , 2009, Nucleic acids research.

[4]  N. Tabata,et al.  Rapid antibody selection by mRNA display on a microfluidic chip , 2009, Nucleic acids research.

[5]  Richard W Roberts,et al.  In vitro selection of protein and peptide libraries using mRNA display. , 2009, Methods in molecular biology.

[6]  Miho Suzuki,et al.  An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution , 2002, Biological Procedures Online.

[7]  Y. Husimi,et al.  Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA , 2006, Nucleic acids research.

[8]  S. Klußmann,et al.  Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist , 2005, Nucleic acids research.

[9]  Andreas Plückthun,et al.  In-vitro protein evolution by ribosome display and mRNA display. , 2004, Journal of immunological methods.

[10]  T. Sawasaki,et al.  Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins. , 2003, Nucleic acids research.

[11]  George Georgiou,et al.  Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer. , 2002, Nucleic acids research.

[12]  Y. Husimi,et al.  An in vitro DNA virus for in vitro protein evolution , 2001, FEBS letters.

[13]  P. Lohse,et al.  cDNA–Protein Fusions: Covalent Protein–Gene Conjugates for the In Vitro Selection of Peptides and Proteins , 2001 .

[14]  L. Gold,et al.  mRNA display: Diversity matters during in vitro selection , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  P. Lohse,et al.  Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions. , 2000, Nucleic acids research.

[16]  M. Taussig,et al.  Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. , 1997, Nucleic acids research.

[17]  J W Szostak,et al.  RNA-peptide fusions for the in vitro selection of peptides and proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Y Husimi,et al.  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 , 1997, FEBS letters.

[19]  Y. Kinoshita,et al.  Restriction-Enzyme-Nondependent Recombination and Rearrangement of DNA (RRR) , 1995 .

[20]  R. Kaptein,et al.  Solution structure of the POU-specific DNA-binding domain of Oct-1 , 1993, Nature.

[21]  I. Shimada,et al.  Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures. , 1992, Biochemistry.