Site‐Specific Fluorescent and Affinity Labelling of RNA by Using a Small Engineered Twin Ribozyme

Over the past decade, RNA has become a focus of investigation into structure–function relationships. A large number of methods for structural studies of RNA are available. Application of those techniques often requires decoration of the sample with reporter groups and modifications such as fluorophores, cross-linking reagents, phosphorothioates, affinity tags or ESR spin labels, most desirably at a specific position. We have developed a strategy for RNA modification that relies on a small engineered twin ribozyme that mediates the exchange of a patch of residing sequence of substrate RNA with a separately added synthetic RNA fragment. Here we show that RNA fragments conjugated with fluorescent dyes or biotin are well accepted for strand exchange. Up to 53% of a dye-labelled oligoribonucleotide has been inserted into a 145-mer RNA. Thus, for the first time, specific labelling of a long transcribed RNA at an internal predetermined position is demonstrated. Modified nucleosides can be site-specifically incorporated into RNA by chemical synthesis with modified nucleoside phosphoramidites. While this is a useful strategy for modification of synthetically available RNAs, modification of long transcripts or natural RNA requires alternative techniques. In this case, specific labelling is possible at the two ends by taking advantage of the unique reactivity of the RNA termini. Functionalization at internal sites can be achieved by adding modified nucleoside triphosphates to the transcription mixture. However, the range of modified nucleosides that can be incorporated during transcription is limited by the specificity of the polymerase, and the label becomes distributed over the entire molecule. The recently published procedure of indirect labelling through oligonucleotide hybridization is a useful alternative. However, it is restricted by the availability of specific hybridization sites in the folded state of the molecule. We have developed a procedure for manipulating at will a chosen patch of a given RNA sequence. A small engineered twin ribozyme promotes, in a strictly controlled fashion, two RNA-cleavage events and two ligations, and thus mediates the specific exchange of RNA patches. The strategy relies on the cleavage/ligation characteristics of the hairpin ribozyme, a small naturally occurring catalytic RNA. Twin ribozymes are derived from tandem duplication of the hairpin ribozyme and thus inherit cleavage as well as ligation activity. Efficient fragment exchange is achieved by destabilization of the duplex between the ribozyme and the RNA patch to be removed (dark grey sequence (lower case letters) in Scheme 1a) and stabilization of the duplex between ribozyme and fragment to be inserted (light grey sequence). After cleavage, the sequence patch (in lower case letters) is readily released from the ribozyme–substrate complex due to the GAUU tetraloop designed to weaken its binding. The new fragment (light grey) contains the four additional nucleotides complementary to the GAUU tetraloop and thus, upon binding into the gap left by dissociation of the cleaved out fragment (dark grey lower case letters), extends the formerly interrupted duplex to 12 base pairs and makes it contiguous. Thus, the structure of the substrate–ribozyme complex is stabilized, and, consequently, ligation of the new fragment to the remaining substrate arms is favoured. In previous experiments, up to 30% of substrate RNA have been transferred into the insertion product. We decided to exploit this strategy in a scheme of site-directed RNA functionalization. To this end, a uridine in the centre of the insertion fragment was replaced by an aminomodified deoxythymidine (marked T in Scheme 1, insertion fragment RS-TW5-6). The aliphatic amino group within the RNA chain was treated with either the isothiocyanate (RS-TW57 and RS-TW5-8) or O-succinimidyl ester derivatives (RS-TW5-9 and RS-TW5-10) of a number of dyes as well as biotin to deliver the modified oligonucleotides shown in Scheme 1b. The fragment-exchange reaction was carried out by using the unlabelled oligonucleotide RS-TW5-6 as well as the four oligonucleotides to which either fluorescein (RS-TW5-7), tetramethylrhodamine (RS-TW5-8), Cy5 (RS-TW5-9) or biotin (RS-TW5-10) had been conjugated to replace the defined patch (lower case letters) of the RNA substrate S-TW5-1. During reaction, the 45[a] S. Vaul on, Dr. S. A. Ivanov, S. Gwiazda, Prof. Dr. S. M ller Ruhr-Universit! t Bochum, Fakult! t Chemie Universit! tsstraße 150, 44780 Bochum (Germany) Fax: (+49)234-321-4783 E-mail : sabine.w. mueller@rub.de Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.