During the past decades, RNA has emerged as a major player in most cellular processes.Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA were in vitro syntheses.Here, we describe a generic approach for expressing and purifying structured RNA in E.coli, using a series of tools which parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the "tRNA scaffold", in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular ribonucleases. This opens the way to large scale structural and molecular investigations of RNA function. IntroductionSystematic studies of protein structure, function and interactions such as structural genomics and double-hybrid approaches have greatly benefited from the development of efficient recombinant expression systems. Simultaneously, new roles have been evidenced for structured RNA in translation, chromosome maintenance, viral replication, as riboswitches or ribozymes [1][2][3] . As a result, these RNA are considered as promising drug targets, as for instance bacterial rRNA (antibiotics) or telomerase RNA 4 . Owing to their large structural repertoire, folded RNA are also interesting molecules for constructing self-assembling nano-objects, some of which have been used for delivering therapeutic siRNA 5,6 . As opposed to proteins, systematic biochemical, structural and pharmacological studies of RNA have however been hampered by difficulties in obtaining homogeneous samples in large quantities. So far, RNA has mostly been produced in vitro using either T7 RNA polymerase transcription 7 or chemical synthesis 8 , which are costly and cumbersome. Producing recombinant RNA in vivo could be an alternative, but thus far has been plagued by a number of obstacles, such as heterogeneity of the products, degradation by ribonucleases and difficulty in purifying the RNA from cell extracts. For example, RNA transcripts overexpressed in vivo using the T7 system 9 are long and heterogeneous, as a consequence of terminator read-through, and have never been 2 purified. 5S ribosomal RNA 10 and tRNA [11][12][13][14] are two exceptions which have been successfully expressed in E. coli. In the latter case, it works because tRNA are recognized by cellular enzymes that precisely process the primary transcript, incorporate modified nucleotides and repair their 3'-end 15 . This results in a single product of defined length, a key feature for structural studies. Furthermore, tRNA adopt a compact 3D structure which makes them resistant to both unfolding and nucleases.We used this robustness and precise processing to express other structured RNA in vivo and designed a generic method using tRNA as a protective scaffold. We extracted the desired recombinant RNA with high yield and purified it to homogeneity using standard chromatography. We employ tools ...
RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes a transfer RNA (tRNA) as a scaffold that can be removed from the RNA of interest by digestion of the fusion RNA at a designed site by RNase H. The tRNA scaffold serves to enhance the stability and to promote the proper expression of its fusion partners. This protocol describes how to construct a tRNA fusion RNA expression vector; to conduct a pilot experiment to assess the yield of the recombinant RNA both before and after processing of the fusion RNA by RNase H; and to purify the target RNA on a large scale for structural or functional studies. This protocol greatly facilitates production of RNA in a time frame of approximately 3 weeks from design to purification. As compared with in vitro methods (transcription, chemical synthesis), this approach is simple, cheap and well suited for large-scale expression and isotope labeling.
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