Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Srisawat, Chatchawan

doi:10.1093/nar/29.2.e4

Cited by 56 publications

(34 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to test the function of these recombinant molecules, we first analyzed a number of ligand-binding RNAs, such as the aptamer selected against sephadex 21 ( Supplementary Fig. 2).…”

Section: Applicability Of the Trna Scaffold Approachmentioning

confidence: 99%

Recombinant RNA technology: the tRNA scaffold

2007

View full text Add to dashboard Cite

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 ...

show abstract

“…In order to test the function of these recombinant molecules, we first analyzed a number of ligand-binding RNAs, such as the aptamer selected against sephadex 21 ( Supplementary Fig. 2).…”

Section: Applicability Of the Trna Scaffold Approachmentioning

confidence: 99%

Recombinant RNA technology: the tRNA scaffold

2007

View full text Add to dashboard Cite

show abstract

“…Affinity tags for protein isolation and detection have proven very useful for a wide variety of research applications; examples include FLAG epitope (Prickett et al+, 1989), glutathione S-transferase (Simons & Vander Jagt, 1981), polyhistidine (Porath et al+, 1975;Porath, 1992), or protein-A tags (Stahl & Nygren, 1997)+ The study of RNAs or ribonucleoproteins (RNPs) could also be facilitated by a highly specific RNA affinity tag that allows a selective recovery of the tagged RNA and all associated proteins under nondenaturing conditions+ A limited number of RNA affinity tags have already been identified (Bachler et al+, 1999;Srisawat & Engelke, 2001), but these tags have limitations in terms of recovering native RNP complexes in the absence of artificially bound macromolecules+ An alternative approach to the problem is to develop an RNA tag that binds tightly to a commonly available target molecule in such a way that the ligand-RNP complex can be selectively and gently dissociated afterwards+…”

Section: Introductionmentioning

confidence: 99%

Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins

Srisawat

Engelke

2001

RNA

181

184

View full text Add to dashboard Cite

RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.

show abstract

“…To further extend the application of aptamers as RNA affinity tags, our lab has identified two artificially selected RNA aptamers that have compact, defined structures and are particularly useful for the study of RNA-protein complexes formed in vivo and isolated under native conditions (20,21). Their applications to the study of RNA-protein complexes as well as the strengths and weaknesses of each aptamer are described here.…”

Section: Introductionmentioning

confidence: 99%

“…Several factors influenced the selection of the target molecule: (1) availability and price of the potential affinity resins, (2) low background affinity (i.e., the resin did not have a high affinity for nonspecific RNAs), and (3) the complexes could be eluted under native conditions. The two target molecules that met these criteria were streptavidin and dextran B512 (in the insoluble form of Sephadex beads), and aptamers to each of these resins were successfully produced (20,21). The consensus sequence for each of these aptamers is shown in Fig.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

RNA Affinity Tags for the Rapid Purification and Investigation of RNAs and RNA–Protein Complexes

Walker

Scott

Srisawat

et al. 2008

Methods in Molecular Biology

View full text Add to dashboard Cite

SummaryIsolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using affinity purification methods that are specific for the RNA rather than the protein components of these complexes. We describe the use of two such RNA affinity tags: small RNAs that bind with high affinity and specificity to either Sephadex beads or streptavidin affinity resins and can be eluted under mild, native conditions that retain intact complexes. The tags can be inserted into appropriate locations in genes encoding the RNA components, and ribonucleoproteins can be assembled either in vivo or in vitro before affinity isolation. Strategies toward the design and production of these tagged RNA sequences are discussed, and the purification procedure is outlined.

show abstract

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Cited by 56 publications

References 19 publications

Recombinant RNA technology: the tRNA scaffold

Recombinant RNA technology: the tRNA scaffold

Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins

RNA Affinity Tags for the Rapid Purification and Investigation of RNAs and RNA–Protein Complexes

Contact Info

Product

Resources

About