RNA detection using peptide-inserted Renilla luciferase.
A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100 pM) exceeds that of Fluc by approximately two orders of magnitude.
Andou
Takashi
T
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Endoh
Tamaki
T
Mie
Masayasu
M
Kobatake
Eiry
E
D001665:Binding Sites; D015696:Gene Products, tat / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry; D016087:Immunodeficiency Virus, Bovine / Q000737:chemistry; D049410:Luciferases, Renilla / Q000737:chemistry / Q000378:metabolism*; D015336:Molecular Probe Techniques; D010455:Peptides / Q000737:chemistry*; D012313:RNA / Q000032:analysis*; D015347:RNA Probes / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity
D015696:Gene Products, tat; D010455:Peptides; D015347:RNA Probes; D011994:Recombinant Proteins; D012313:RNA; D049410:Luciferases, Renilla