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Recent advances in RNA engineering during the last two decades have supported the development of RNA-based therapeutics targeting a variety of human diseases. The broad scope of these emerging drugs clearly demonstrates the versatility of RNA. Ribozymes have been seen as promising candidates in this area. However, efficient intracellular application of ribozymes remains challenging, and other strategies appear to have outperformed ribozymes as molecular drugs. Nevertheless, trans-cleaving ribozymes have been applied for specific cleavage of target mRNAs in order to inhibit undesired gene expression. Furthermore, ribozymes have been engineered to allow site-directed RNA sequence alterations, enabling the correction of genetic misinformation at the RNA level. This chapter provides an overview of ribozyme-based strategies, highlighting the promises and pitfalls for potential therapeutic applications.

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.

Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry (ITC). The targeted MYC3 sequence lacks 3'-flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt-ended 3'-outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand/quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm ligand-induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3'-outer tetrads. Additional ligand molecules in excess bind to the 5'-outer tetrads of the sandwich complex. Such a ligand-promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex-based technologies.

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 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.

RNA repair is an emerging strategy for gene therapy. Conventional gene therapy typically relies on the addition of the corrected DNA sequence of a defective gene to restore gene function. As an additional option, RNA repair allows alteration of the sequence of endogenous messenger RNAs (mRNAs). mRNA sequence alteration is either facilitated by intracellular spliceosome machinery or by the intrinsic catalytic activity of trans-acting ribozymes. Previously we developed twin ribozymes, derived from the hairpin ribozyme, by tandem duplication and demonstrated their potential for patchwise RNA repair. Herein we describe the development of such a twin ribozyme for potential repair of a deletion mutation in the oncogenic CTNNB1-ΔS45 mRNA. We demonstrate that hairpin ribozyme units within the twin ribozyme can be adapted to efficiently cleave/ligate non-consensus substrates by introduction of compensatory mutations in the ribozyme. Thus, we show the twin ribozyme mediated repair of truncated CTNNB1 transcripts (up to 1000 nt length). Repair of the entire CTNNB1-ΔS45 mRNA, although apparently possible in general, is hampered in vitro by the secondary structure of the transcript.

In the transition from the RNA world to the modern DNA/protein world, RNA-catalyzed aminoacylation might have been a key step towards early translation. A number of ribozymes capable of aminoacylating their own 3' termini have been developed by in vitro selection. However, all of those catalysts require a previously activated amino acid-typically an aminoacyl-AMP-as substrate. Here we present two ribozymes connected by intermolecular base pairing and carrying out the two steps of aminoacylation: ribozyme 1 loads nonactivated phenylalanine onto its phosphorylated 5' terminus, thereby forming a high-energy mixed anhydride. Thereafter, a complex of ribozymes 1 and 2 is formed by intermolecular base pairing, and the "activated" phenylalanine is transferred from the 5' terminus of ribozyme 1 to the 3' terminus of ribozyme 2. This kind of simple RNA aminoacylase complex was engineered from previously selected ribozymes possessing the two required activities. RNA aminoacylation with a nonactivated amino acid as described here is advantageous to RNA world scenarios because initial amino acid activation by an additional reagent (in most cases, ATP) and an additional ribozyme would not be necessary.

Twin ribozymes mediate the exchange of a short patch of RNA against an exogenous oligonucleotide within a suitable RNA substrate. Thus, twin ribozymes are promising tools for RNA repair, i.e. for the treatment of genetic disorders at the mRNA level. A number of twin ribozyme-mediated RNA fragment exchange reactions have been successfully demonstrated using short model substrates. Herein we show for the first time a twin ribozyme-mediated in vitro repair of a full-length transcript and translation into a functional protein. The system is based on the repair of a designed mutant EGFP mRNA containing the four-base deletion ΔACTC (190–193). Upon twin ribozyme-mediated replacement of a patch of 15 nucleotides with an externally added repair oligonucleotide (19 mer) the wild type sequence of the EGFP transcript could be restored with 32% yield. This is the first time that such a high twin ribozyme-mediated repair yield, so far observed only for short model substrates, has been obtained for a full-length mRNA. Translation of the repaired EGFP-ΔACTC mRNA produces functional EGFP, as detected by the restored fluorescence.

Site-specific functionalization of RNA and DNA molecules has become a major task in nucleic acid chemistry. Synthetic RNAs are required for a large number of applications. Beyond synthetic RNA for antisense, aptamer, ribozyme, and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. A wide variety of monomer-building blocks is commercially available to be used for site-specific incorporation by solid-phase RNA synthesis. However, the efficient chemical preparation of RNA is limited to oligomers of about 60–70 nucleotides. Therefore, efficient strategies for fragment ligation or other alternative novel protocols are required. We here provide an overview on our work on the synthesis of site-specifically modified long-mer RNAs, focusing on enzymatic and chemical ligation of synthetic RNA fragments and on ribozyme-mediated sequence exchanges.

Over the past two decades, RNA catalysis has become a major topic of research. On the one hand, naturally occurring ribozymes have been extensively investigated concerning their structure and functional mechanisms. On the other hand, the knowledge gained from these studies has been used to engineer ribozyme variants with novel properties. In addition to RNA engineering by means of rational design, powerful techniques for selection of ribozymes from large pools of random sequences were developed and have been widely used for the generation of functional nucleic acids. RNA as catalyst has been accompanied by DNA, and nowadays a large number of ribozymes and deoxyribozymes are available. The field of ribozyme generation and selection has been extensively reviewed. With respect to the field of biotechnology, RNA and DNA catalysts working on peptides or proteins, or which are designed to control protein synthesis, are of utmost importance and interest. Therefore, in this review, we will focus on engineered nucleic acid catalysts for peptide synthesis and modification as well as for intracellular control of gene expression.

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.