| Popis: |
The structure of an RNA molecule is often critical for its correct functioning, post-transcriptional regulation, and/or translation. Predicting RNA secondary structures with in silico tools is relatively straightforward with the large array of software and webservers available. However, for long RNAs and RNA at high temperatures, in silico predictions are less accurate and require experimental validation. To this end, a variety of structural probing reagents are commonly used, both for in vitro and in vivo mapping of RNA structure. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments make use of a nonbase-specific modifying reagent, acylating all conformationally flexible (mainly single-stranded or unpaired) nucleotides and have been employed both for in vitro and in vivo modification of RNA. Here, we describe a protocol for an in vitro SHAPE experiment, starting from in vitro transcribed RNA using a T7 polymerase system. This RNA is folded and subsequently modified in vitro with the SHAPE-reagent N-methyl isatoic anhydride (NMIA). Primer extension employing a radioactive 32P-labeled primer that binds the RNA downstream of the structure of interest will generate cDNA until the reverse transcriptase enzyme is halted by the introduced SHAPE modifications. Denaturing acrylamide gel electrophoresis of the pool of 32P-labeled cDNAs and the corresponding sequencing ladders, followed by autoradiography, will expose these stops in reverse transcription (RT) and will therefore enable to identify single-stranded nucleotides in the RNA of interest. These RT stops and NMIA-modification efficiencies can be quantified with ImageJ software and can be used to validate or increase the accuracy of RNA secondary structure predictions. |
