Construction of Intramolecular Luciferase Complementation Probe for Detecting Specific RNA

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• 作者：Tamaki Endoh ; Masayasu Mie ; Hisakage Funabashi ; Tatsuya Sawasaki ; Yaeta Endo ; Eiry Kobatake
• 刊名：Bioconjugate Chemistry
• 出版年：2007
• 出版时间：May 2007
• 年：2007
• 卷：18
• 期：3
• 页码：956 - 962
• 全文大小：605K
• 年卷期：v.18,no.3(May 2007)
• ISSN：1520-4812

文摘

Intermolecular enzyme complementation assay is a useful method for detecting protein-protein interactions.Specifically, bioluminescent signals produced from reconstructed split luciferase fragments are powerful toolsfor in vivo analysis because the bioluminescent signals have been visualized both in cultured cells and livinganimals. However, they are limited for detection and evaluation of biological events relevant to intermolecularprotein-protein interactions. In this study, we constructed an intramolecular luciferase complementation probefor detecting target biomolecules other than protein-protein interactions. It consists of peptide-inserted fireflyluciferase (PI-FLuc) containing a short peptide between internally divided firefly luciferase. The inserted shortpeptide triggers FLuc complementation or discomplementation and subsequent reactivation or inactivation ofFLuc activity through its induced fit conformational changes. We chose RNA binding arginine rich motif (ARM)peptides, Rev and/or Tat, for model peptide insertion, and expressed constructed PI-FLuc probe variants using awheat germ cell-free protein synthesis system. They showed FLuc activity changes, reactivation, or inactivationafter binding to their specific RNA targets. Furthermore, to expand the versatility of the PI-FLuc RNA detectionsystem, we designed split-RNA probes built to reform the ARM peptide binding site in the presence of arbitrarilyselected target-RNA. As a result, the target RNA was homogeneously detected by FLuc luminescent signalsmediated by a cooperative function of the PI-FLuc and split-RNA probe sets.