Evaluation of small ligand-protein interactions by using T7 RNA polymerase with DNA-modified ligand.
The interaction between proteins and ligands was evaluated by T7 RNA polymerase transcription with a DNA-modified ligand. The principle of this method is suppression of T7 RNA polymerase transcription by binding of a protein to small ligand modified by conjugation with a T7 RNA polymerase promoter. To demonstrate proof of principle, biotin or antifolate methotrexate was modified by covalent attachment of a T7 RNA promoter. Using these T7 RNA promoter-modified ligands, T7 RNA polymerase transcriptions were performed in the presence or absence of an anti-biotin antibody or recombinant human dihydrofolate reductase, respectively. Transcription was suppressed in the presence of each binding protein plus its modified ligand, but not in the absence of the binding protein.
Mie
Masayasu
M
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
Sugita
Rie
R
Endoh
Tamaki
T
Kobatake
Eiry
E
D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001710:Biotin / Q000276:immunology; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D008024:Ligands; D011994:Recombinant Proteins; D014764:Viral Proteins; D001710:Biotin; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases