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 ...

No abstract

Electrochemical impedance spectroscopy is successfully utilized for label-free monitoring of the cleavage reaction of a RNA substrate by a complementary hairpin ribozyme. The formation of the RNA substrate/ribozyme complex is increasing the negative charge at the interface and is modulating the kinetic of the redox conversion of a negatively charged redox mediator (e.g. [Fe(CN) 6 ] 3À/4À ) hence leading to an increase in the charge transfer resistance. Upon addition of bivalent cations such as Mg 2 + ions the conformation of the ribozyme is changed and the catalytic cleavage of the RNA is monitored by a decrease of the charge transfer resistance.