Laser-mediated, site-specific inactivation of RNA transcripts
Laser-mediated, site-specific inactivation of RNA transcripts
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DOI：
10.1073/pnas.96.11.6131
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发表时间：
1999-05-25
影响因子：
11.1
通讯作者：
Wilson, C
中科院分区：
文献类型：
--
作者：
Grate, D;Wilson, C
特定基因产物的生物学功能通常是通过阻断其在生物体中的表达并观察其产生的表型来确定的。使用孔雀石绿（MG）标记抗体的发色团辅助激光灭活使得以高度受限的方式灭活靶蛋白成为可能，探测其时间和空间分辨功能。在本报告中，我们描述了MG结合RNA基序的分离和体外表征，该基序可以在RNA水平（RNA-发色团辅助激光失活）上对基因功能进行相同的高分辨率分析。一个定义明确的不对称内部凸起在RNA双工允许高亲和力和高特异性结合的MG。低浓度MG存在下的激光照射可诱导MG结合RNA的破坏，但不能破坏共孵育的对照RNA。激光诱导的mg结合RNA的水解主要局限于凸起内的单个核苷酸。通过适当地将该基序整合到目标基因中，基因产生的转录本可以有效地标记为激光介导的破坏。
The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite 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 irt vitro characterization 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.