RNA self-splicing by engineered hairpin ribozyme variants.

Small RNAs capable of self-cleavage and ligation might have been the precursors for the much more complex self-splicing group I and II introns in an early RNA world. Here, we demonstrate the activity of engineered hairpin ribozyme variants, which as self-splicing introns are removed from their parent RNA. In the process, two cleavage reactions are supported at the two intron-exon junctions, followed by ligation of the two generated exon fragments. As a result, the hairpin ribozyme, here acting as the self-splicing intron, is cut out. Two self-splicing hairpin ribozyme variants were investigated, one designed by hand, the other by a computer-aided approach. Both variants perform self-splicing, generating a cut-out intron and ligated exons.

A Hairpin Ribozyme Derived Spliceozyme.

The vast majority of RNA splicing in today's organisms is achieved by the highly regulated and precise removal of introns from pre-mRNAs via the spliceosome. Here we present a model of how RNA splicing may have occurred in earlier life forms. We have designed a hairpin ribozyme derived spliceozyme that mediates two RNA cleavages and one ligation event at specific positions and thus cuts a segment (intron) out of a parent RNA and ligates the remaining fragments (exons). The cut-out intron then performs a downstream function, acting as a positive regulator of the activity of a bipartite DNAzyme. This simple scenario shows how small RNAs can perform complex RNA processing dynamics, involving the generation of new phenotypes by restructuring segments of given RNA species, as well as delivering small RNAs that may play a functional role in downstream processes.

Preparation of modified long-mer RNAs and analysis of FMN binding to the ypaA aptamer from B. subtilis.

In recent years, RNA has been shown to fulfil a number of cellular functions. This has led to much interest in elucidation of the structure of functional RNA molecules, and thus, in the preparation of suitably functionalized RNAs. The chemical synthesis of RNAs allows for the site-specific modification; however, is limited to sequences of about 60-70 nucleotides in length. At the example of the flavine mononucleotide (FMN) responsive aptamer of the ypaA riboswitch from B. subtilis, we demonstrate the highly efficient preparation of site-specifically modified long-mer RNAs. Our strategy consists of the chemical synthesis of fragments followed by enzymatic or chemical ligation. Splint ligation with T4 RNA ligase turned out to be most successful among the enyzymatic protocols. Highly efficient chemical ligation was performed by azide-alkyne cycloaddition of suitably modified RNA fragments. Wild-type and 2-aminopurine (2-AP)-modified variants of the ypaA aptamer were prepared. FMN binding to all synthesized ypaA aptamer variants is demonstrated. However, dissociation of FMN from its binding site by reduction of the isoalloxazin unit as demonstrated before for a small-hairpin-derived aptazyme could not be shown. This implies that either FMN is less accessible to reduction when it is bound to its natural aptamer; that reduced FMN remains bound to the aptamer; or that FMN upon reduction indeed is released from its binding site, without the aptamer folding back in the natural ligand-free state. The results of this study are of general interest to the preparation of site-specifically modified RNAs for investigation into structure and function.

Challenges and Perspectives in Nucleic Acid Enzyme Engineering.

Engineering of nucleic acids has been a goal in research for many years. Since the discovery of catalytic nucleic acids (ribozymes and DNAzymes), this field has attracted even more attention. One reason for the increased interest is that a large number of ribozymes have been engineered that catalyze a broad range of reactions of relevance to the origin of life. Another reason is that the structures of ribozymes or DNAzymes have been modulated such that activity is dependent on allosteric regulation by an external cofactor. Such constructs have great potential for application as biosensors in medicinal or environmental diagnostics, and as molecular tools for control of cellular processes. In addition to the development of nucleic acid enzymes by in vitro selection, rational design is a powerful strategy for the engineering of ribozymes or DNAzymes with tailored features. The structures and mechanisms of a large number of nucleic acid catalysts are now well understood. Therefore, specific design of their functional properties by structural modulation is a good option for the development of custom-made molecular tools. For rational design, several parameters have to be considered, and a number of tools are available to help/guide sequence design. Here, we discuss sequence, structural and functional design using the example of hairpin ribozyme variants to highlight the challenges and opportunities of rational nucleic enzyme engineering.

Engineering of hairpin ribozyme variants for RNA recombination and splicing.

The hairpin ribozyme is a small, naturally occurring RNA that catalyzes the reversible cleavage of RNA substrates. Among the small endonucleolytic ribozymes, the hairpin ribozyme possesses the unique feature of the internal equilibrium between cleavage and ligation being shifted toward ligation. This allows control of the reaction outcome by structural design: fragments that are strongly bound to the ribozyme are preferentially ligated, whereas substrates that easily dissociate upon cleavage, such that they are not available for religation, are preferentially cleaved. We have made use of this characteristic feature in engineering a number of hairpin ribozyme variants by programmed conformational design that carry out cascades of cleavage and ligation reactions, and as a result mediate more complex RNA processing reactions. Here, we review our work on the engineering of hairpin ribozyme variants for RNA recombination and regular and back-splicing, and discuss the relevance of such activities in early life.

Hairpin ribozyme mediated RNA recombination.

We have engineered a hairpin ribozyme that in a two-step reaction supports RNA recombination by catalysing the cleavage of two non-functional RNA precursors (first step) followed by the recombination of two of the cleavage fragments into a functional RNA (second step). The recombination product (yield: 76%) is a fully functional hammerhead ribozyme.

Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today?

Since the discovery of the first catalytic RNA in 1981, the field of ribozyme research has developed from the discovery of catalytic RNA motifs in nature and the elucidation of their structures and catalytic mechanisms, into a field of engineering and design towards application in diagnostics, molecular biology and medicine. Owing to the development of powerful protocols for selection of nucleic acid catalysts with a desired functionality from random libraries, the spectrum of nucleic acid supported reactions has greatly enlarged, and importantly, ribozymes have been accompanied by DNAzymes. Current areas of research are the engineering of allosteric ribozymes for artificial regulation of gene expression, the design of ribozymes and DNAzymes for medicinal and environmental diagnostics, and the demonstration of RNA world relevant ribozyme activities. In addition, new catalytic motifs or novel genomic locations of known motifs continue to be discovered in all branches of life by the help of high-throughput bioinformatic approaches. Understanding the biological role of the catalytic RNA motifs widely distributed in diverse genetic contexts belongs to the big challenges of future RNA research.