Novel approach for linking genotype to phenotype in vitro by exploiting an extremely strong interaction between RNA and protein.
We recently isolated an aptamer that binds to the Tat protein of HIV-1 with extremely high affinity and specificity (Yamamoto, R.; et al. Genes Cells 2000, 5, 371.). In the present study, we exploited this strong binding to develop a novel coupling method that links genotype with phenotype. To strengthen the original RNA-protein interaction still further, we connected three units of the aptamer in tandem and three units of a peptide derived from Tat that interacted with the aptamer. The binding of the resultant RNA, which consisted of three units of the aptamer, to the resultant peptide, which consisted of three units of the peptide, was extremely strong. In fact, the RNA-protein interaction was one of the strongest ever reported, with an apparent K(d) below 16 pM. This strong interaction was attempted for the selection of functional proteins, namely, dihydrofolate reductase (DHFR) or streptavidin, which we chose as an example, and we succeeded in the expected selection, although to a limited extent, of the target protein. The noncovalent but strong interaction described above should be useful as a novel tool for the future selection of functional proteins from pools of random sequences of amino acids.
Fujita
Satoshi
S
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
Sawata
Shinya Y
SY
Yamamoto-Fujita
Rika
R
Endo
Yaeta
Y
Kise
Hideo
H
Iwakura
Masahiro
M
Taira
Kazunari
K
D018388:Codon, Terminator; D004591:Electrophoresis, Polyacrylamide Gel; D015696:Gene Products, tat / Q000737:chemistry; D005838:Genotype; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
D018388:Codon, Terminator; D015696:Gene Products, tat; D010455:Peptides; D011506:Proteins; D012333:RNA, Messenger; D012313:RNA; D019809:Streptavidin; D013762:Tetrahydrofolate Dehydrogenase