Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins.
In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.
Doi
Nobuhide
N
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
Takashima
Hideaki
H
Wada
Aiko
A
Oishi
Yuko
Y
Nagano
Tetsuya
T
Yanagawa
Hiroshi
H
D000345:Affinity Labels / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000528:radiation effects; D015723:Gene Library; D005838:Genotype; D008027:Light; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D019151:Peptide Library; D010777:Photochemistry / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D009696:Nucleic Acids; D019151:Peptide Library; D011994:Recombinant Proteins