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.[1] 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.[2] 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.[3] Functionalization at internal sites can be achieved by adding modified nucleoside triphosphates to the transcription mixture. [4] 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.[5]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.[2] The strategy relies on the cleavage/ligation characteristics of the hairpin ribozyme, [6] 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 1 a) 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 ...

Reversible chemistry, allowing for chain-forming as well as chain-breaking steps, is important for biological self-organization. In this context, ribozymes, catalyzing both RNA cleavage and ligation, may have significantly contributed to extending the sequence space and length of RNA molecules in early life forms. Here we present an engineered RNA that self-processes by passing through a number of cleavage and ligation steps. Intermolecular reactions compete with intramolecular reactions, resulting in a variety of products. Our results demonstrate that RNA can undergo self-oligomerization, which may have been important for extending the RNA genome size in RNA world scenarios.

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In recent years RNA has become the focus of development into new diagnostic and therapeutic schemes. Antisense-RNA, ribozyme, aptamer and siRNA technologies have been developed and have found application in molecular medicine [1][2][3][4][5][6][7]. Signalling aptamers and aptazymes have been constructed that can sense a number of molecules in real time and thus are valuable diagnostic tools [8][9][10]. Furthermore, recently discovered riboswitches that regulate gene expression in vivo in response to specific metabolites [11][12][13] or temperature [14] may lead to new RNA-based therapeutic strategies.Elucidation of the molecular principles of RNA functioning in a specific context has led to the engineering of RNA molecules with new functions. Two complementary strategies can be used in RNA engineering: rational design and directed evolution. Whereas directed molecular evolution relies on the creation of a repertoire of modified RNAs from which beneficial variants are filtered, in a rational design experiment, defined changes in the nucleotide sequence and ⁄ or secondary structure of a specific RNA are planned on the basis of a preconceived idea. This requires detailed structural and mechanistic information on the parent RNA. In cases where this information is available, rational design has contributed to the development of new functional RNA, for example, signalling aptamers and aptazymes [8][9][10].Work in our laboratory has focused on the rational design of functional RNA, in particular on the development of hairpin-derived twin ribozymes for site-specific alteration of RNA sequences, and fluorescent and affinity labelling of large RNA molecules [15][16][17][18]. The hairpin ribozyme catalyses the reversible site-specific cleavage of suitable RNA substrates, generating fragments with a 2¢,3¢-cyclic phosphate and, respectively, a free 5¢-OH terminus [19,20]. In the reverse reaction, the oxygen atom of the free 5¢-OH group of one RNA fragment attacks the phosphorous of the cyclic 2¢,3¢-phosphate group of another, resulting in ligation of the two fragments. In contrast to the hammerhead ribozyme, the conformation of the hairpin ribozyme-substrate complex does not change significantly upon cleavage: the two cleavage fragments In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into useful tools. Work in our laboratory has focused on the development of twin ribozymes for sitespecific RNA sequence alteration. To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation. The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min )1 and a final yield of 70%. We combined a reverse-joined hairpin ribozyme with...

The hairpin ribozyme belongs to the class of self-cleaving nucleases that are found in plant viroids, virussoids or viral satellite RNAs. [1] The 50-nucleotide-long minimal sequence (Figure 1) catalyses the reversible specific cleavage of a suitable 14nucleotide-long RNA substrate. The secondary structure of the Received: September 19, 2002 [Z 491] [a] Dr.