Andou Takashi, Endoh Tamaki, Mie Masayasu, Kobatake Eiry
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Anal Bioanal Chem. 2009 Jan;393(2):661-8. doi: 10.1007/s00216-008-2473-2. Epub 2008 Nov 1.
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.
构建了一种新型的互补系统，该系统包含短肽插入的海肾荧光素酶（PI-Rluc）和分裂RNA探针，用于无创RNA检测。RNA结合肽HIV-1 Rev和BIV Tat用作插入肽。它们在与特定RNA结合时会发生诱导契合构象变化，并触发Rluc的互补或反互补。分裂RNA探针被设计为在与任意选择的靶RNA杂交时重新形成肽结合位点。这组重组蛋白和分裂RNA探针在RNA检测中具有高度敏感性。在本研究中，我们表明Rluc系统与Fluc相当，但其对任意选择的RNA的检测限（至少100 pM）比Fluc高出约两个数量级。
