Laser-mediated, site-specific inactivation of RNA transcripts

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 ...

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“…4b). We obtained similar results with 5 aptamers selected against streptavidin 22 or malachite green 23 (Supplementary Fig. 3).…”

Section: Applicability Of the Trna Scaffold Approachsupporting

confidence: 67%

Recombinant RNA technology: the tRNA scaffold

2007

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“…In 1999, Grate et al developed a 38-mer RNA aptamer (see Fig. 2) that bound to MG with a dissociation constant (K d ) of less than 1μM (Grate & Wilson, 1999). The K d value for the aptamer produced for use in this study was estimated to be approximately 600 nM, assuming a 1:1 binding model.…”

Section: Fluorescence-based Detection Of Malachite Green In Fish Tissuementioning

confidence: 92%

“…Jiang et al, 2011) Pesticides Herbicides Atrazine DNA 2000 -- (Sinha et al, 2010) Fungicides RNA <1000 -- (Grate & Wilson, 1999) Malachite Green 600 Fluorescence - (Stead et al, 2010) Inorganic ions Arsenic (3 + , 5 + )D N A 5 ( 3 + ), 7 (5 + ) Colorimetric 0.6 nM Resonance Scattering 34 fg/mL (Z. Jiang et al, 2010) Mercury (2 + ) DNAFluorescence 220 µM …”

Section: Food Packagingmentioning

confidence: 99%

“…Scientific evidence indicates that a metabolite of malachite green (LMG), leucomalachite green, may be a genotoxic carcinogen that persists in fish tissues. A malachite green aptamer was developed in 1999 (Grate & Wilson, 1999). This aptamer has been recently used by Stead et al as the recognition element in a fluorescence-based screening assay for MG detection in fish tissue (Stead et al, 2010).…”

Section: Pesticidesmentioning

confidence: 99%

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Advances in Aptamer-Based Biosensors for Food Safety

McKeague¹,

Giamberardino²,

DeRosa³

2011

Environmental Biosensors

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“…Liao et al exploited this property in 1994 by conjugating MG to antibodies to target proteins in vivo, a technique called " Chromophore-assisted laser inactivation" (Liao et al, 1994). This inspired Grate and Wilson to select an RNA aptamer to bind MG and selectively target specific RNA molecules for inactivation in vivo (Grate & Wilson, 1999). An RNA aptamer to Hoechst dye 33258 was also selected to study gene regulation while RNA aptamers for Sulforhodamine B and Fluorescein were selected for potential in vivo RNA labeling (Werstuck & Green, 1998;Holeman et al, 1998).…”

Section: Fluorogen-binding Aptamer-based Systems With Light-up Propermentioning

confidence: 99%