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Nucleic Acid Enzymes Roberto Fiammengo and Andres Ja¨ Schke

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Nucleic Acid Enzymes Roberto Fiammengo and Andres Ja¨ Schke Nucleic acid enzymes Roberto Fiammengo and Andres Ja¨ schke Since the discovery of the first natural ribozyme more than 20 extensively reviewed [2,4–8] and will not be further years ago, it has become clear that nucleic acids are not only considered here. Moreover, besides the pure scientific the static depository of genetic information, but also possess interest, it should not be forgotten that nucleic acid intriguing catalytic activity. The number of reactions catalyzed enzymes are currently and actively studied as potential by engineered nucleic acid enzymes is growing continuously. molecular therapeutics. These studies are, at least in The versatility of these catalysts supports the idea of an some cases, at such an advanced stage that phase I and ancestral world based on RNA predating the emergence of II clinical trials are underway [9–11]. proteins, and also drives many studies towards practical applications for nucleic acid enzymes. This article aims to highlight developments in the field of artificial nucleic acid enzymes in the past two years. New Addresses catalytic activities have been discovered for both ribo- Institute of Pharmacy and Molecular Biotechnology, University of zymes and DNAzymes. Several studies have expanded Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany the scope and applicability of previously selected nucleic Corresponding author: Ja¨ schke, Andres ([email protected]) acid enzymes or have tried to elucidate the mechanism used to support catalytic activity. Allosterically regulated ribozymes will also briefly be considered; these artificial Current Opinion in Biotechnology 2005, 16:614–621 systems actually predate the discovery of natural ribos- This review comes from a themed issue on witches, with catalytic activity possibly modulated Chemical biotechnology through metabolite–RNA binding. Edited by Peter N Golyshin Non-natural ribozymes Available online 27th October 2005 Despite the lack of chemical diversity characterizing the 0958-1669/$ – see front matter array of functional groups present in RNA, relative to # 2005 Elsevier Ltd. All rights reserved. proteins, ribozymes with unprecedented catalytic activ- ities are continuously being discovered by means of in DOI 10.1016/j.copbio.2005.10.006 vitro selection approaches. These studies are especially relevant in the context of validating the ‘RNA world’ hypothesis [12], but may also have consequences for the Introduction development of novel biotechnological processes. For The term ‘nucleic acid enzyme’ is used to identify nucleic example, nucleic acid catalysts developed for a practically acids that have catalytic activity. Ribozymes (literally relevant organic transformation could be immobilized on enzymes made of ribonucleic acid or RNA) are found solid supports [13], in analogy to current technologies for in nature and mediate phosphodiester bond cleavage and immobilized enzymes [14]. formation and peptide bond formation. Artificial ribo- zymes have been obtained by means of combinatorial Ribozymes showing redox activity have been developed chemistry approaches, such as in vitro selection and in in Suga’s laboratory [15,16]. An alcohol dehydrogenase vitro evolution [1], and have been shown to catalyze quite ribozyme was selected in the presence of NAD+ and Zn2+ a broad array of other chemical reactions [2,3]. Deoxyr- and was found to oxidize a tethered benzyl alcohol ibozymes or DNAzymes (enzymes made of DNA) are substrate to the corresponding aldehyde in a strict cofac- artificial molecules and are not found in nature. tor-dependent fashion [15]. Additionally, one represen- tative clone obtained from this in vitro selection was later Although nucleic acids enzymes are still considered to act found to catalyze the reverse reaction as well [16]. The ‘slowly’ compared with their proteinaceous counterparts, appended benzaldehyde derivative could be reduced to they are often a lot smaller, readily available and easier to the corresponding alcohol in the presence of NADH and study so that many details concerning their catalytic and Zn2+, demonstrating for the first time that ribozymes can molecular recognition mechanisms can be unravelled. sustain reversible redox chemistry. Although the discovery of natural ribozymes dates back more than two decades, questions like ‘How do natural Eaton’s group [17] has reported the selection of a ribo- ribozymes achieve catalysis?’ and ‘To what extent can zyme that promotes the formation of a urea bond between their catalytic mechanisms be compared with those of peptide phosphonate substrates and the exocyclic amino protein enzymes?’ still burn in the scientific community. group of the 30-terminal cytidine residue of the ribozyme. The vast body of research in this field has been recently These particular substrates were employed with the aim Current Opinion in Biotechnology 2005, 16:614–621 www.sciencedirect.com Nucleic acid enzymes Fiammengo and Ja¨ schke 615 of directly influencing the ribozyme’s molecular recogni- ing/deblocking strategy. After seven rounds of selection tion ability for substrates with differences at a distal site aimed at the isolation of short functional ribozymes, the (away from the actual reactive group). An unusual selec- mean pool length was decreased from 163 to 131 nucleo- tion strategy was therefore designed to isolate the active tides with a net deletion frequency within the variable- nucleic acid sequences, that is those catalyzing conjuga- length regions of 41%. tion of the substrate to RNA via urea bond formation. The peptide–RNA conjugates were captured with human The second strategy was applied to family B of the 4SU neutrophile elastase, taking advantage of the known synthase ribozyme and is based on nonhomologous or activity of peptide phosphonate as a suicide inhibitor random recombination [21]. Double-stranded DNA cor- for this enzyme (Figure 1). The selected catalysts med- responding to the sequence of a previously isolated iate urea bond formation at the N terminus of the pep- ribozyme was partially digested with DNase I, and sticky tides and differentiate between substrates with the ends were filled using T4 DNA polymerase. The blunt- opposite configuration to the C-terminal residue. end fragments were then reassembled into new mole- cules that had a broad sequence length distribution by Ribozymes able to synthesize purine nucleotides have reaction with T4 DNA ligase. PCR allowed selection and been selected [18]. Together with the already known amplification of all molecules that had the 50- and 30- ability of RNA to catalyze the synthesis of pyrimidine primer sequences at the corresponding end (108 DNA nucleotides [19], the results reported by the Unrau group sequences), irrespective of internal deletions, inversions [18] show that RNA is able to synthesize all the building and translocations. After size-dependent in vitro selec- blocks from which it is constituted. The same group has tion, the original 271-nucleotide-long ribozyme was also reported two methodological studies aimed at solving reduced to sequences as short as 81 nucleotides. the problem of identifying a ribozyme’s core motif [20,21]. Extraneous sequences found in loops or beyond RNA is not only able to synthesize its building blocks, but the 50 and the 30 boundaries of a ribozyme and unneces- can also catalyze a templated primer extension reaction sary for catalytic activity are easily recognizable and analogously to polymerase enzymes [22]. A novel strategy removable. By contrast, it may prove extremely difficult was developed to measure the processivity of a polymer- to shorten interhelical joining regions by rational design, ase ribozyme showing that — despite its inefficiency — even when these sequences are poorly conserved, indi- the ribozyme is undoubtedly partially processive [23]. cating a secondary role in catalysis. Joyce and coworkers [24] showed that a self-replicating ribozyme could be converted to a cross-catalytic replica- Each of the two reported strategies was applied to one of tion system in which two ribozymes catalyze each other’s the three 4SU synthase ribozyme families previously synthesis from four component substrates [24] identified [19]. Characterization of the core motif of (Figure 2). family A was achieved by the construction of large libraries of deletion and mutation variants with as little Two papers were concerned with ribozymes catalyzing sequence bias as possible [20]. The best way to achieve aminoacylation of RNA substrates [25,26]. This function balanced levels of deletion proved to be a partial reblock- is nowadays carried out by aminoacyl-tRNA synthetase Figure 1 RNA-catalyzed urea-bond formation. (a) Two peptide phosphonate substrates used for the selection of stereoselective urea synthase ribozymes. The reactive group is shown in red and the distal phosphonate group (responsible for the suicide inhibition of neutrophile elastase during selection) in cyan. Note the different configuration of the carbon atom attached to the phosphorous, three peptide bonds away from the reactive site. (b) The selected ribozyme only catalyzes the formation of a urea bond (in green) with a substrate having the correct stereochemistry. www.sciencedirect.com Current Opinion in Biotechnology 2005, 16:614–621 616 Chemical biotechnology Figure 2 Cross-catalytic replication of a ligase ribozyme. Ribozymes T and T0 selectively catalyze the ligation of substrates A0 with B0 and A with B, respectively. Dissociation of the product complex TT0 generates new free copies of the two ribozymes
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