Recombinant RNA technology: the tRNA scaffold

Abstract

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 ...

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

“…It is known that RNA molecule is unstable in E. coli cells due to the rapid degradation caused by endogenous RNases. However, previous studies have shown that tRNA scaffolds can protect RNA from degradation (Normanly et al 1986;Ponchon and Dardel 2007). Taking this into consideration, we made three different structural constructs with Spinach, which are illustrated in Fig.…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…A third method used to produce RNA for NMR studies was recently developed by Dardel and coworkers by producing RNA in vivo using a tRNA scaffold to protect the RNA from cellular RNases [121,122]. The tRNA scaffold can be removed either by DNAzymes or by sequence-specific RNase H cleavage [115,[117][118][119].…”

From DNA nanotechnology to synthetic biology

“…One of the problems for in vivo RNA nanoconstruction is the rapid degradation of RNA. However, recent work suggests that degradation can be avoided by incorporating tRNA structures into the transcripts (Ponchon and Dardel, 2007).…”

“…A similar approach is conceivable for other RNA-based nanodevices. As for in vivo production of RNAbased nanostructures, the tRNA-technique mentioned above may be of great use (Ponchon and Dardel, 2007). In general, however, it may not be straightforward to transfer DNAbased in vitro technology to RNA-based in vivo systems.…”