Laser-mediated, site-specific inactivation of RNA transcripts

摘要

The biological function of specific gene prod- ucts often is determined experimentally by blocking their expression in an organism and observing the resulting phe- notype. Chromophore-assisted laser inactivation using mal- achite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and in vitro character- ization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction.