Nucleic acid enzymes Roberto Fiammengo and Andres Jäschke

Nucleic acid enzymes Roberto Fiammengo and Andres Jäschke Since the discovery of the first natural ribozyme more than 20 years ago, it has become clear that nucleic acids are not only the static depository of genetic information, but also possess intriguing catalytic activity. The number of reactions catalyzed by engineered nucleic acid enzymes is growing continuously. The versatility of these catalysts supports the idea of an ancestral world based on RNA predating the emergence of proteins, and also drives many studies towards practical applications for nucleic acid enzymes. Addresses Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany Corresponding author: Jäschke, Andres (jaeschke@uni-hd.de) This review comes from a themed issue on Chemical biotechnology Edited by Peter N Golyshin Available online 27th October 2005 0958-1669/$ see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.10.006 Introduction The term nucleic acid enzyme is used to identify nucleic acids that have catalytic activity. Ribozymes (literally enzymes made of ribonucleic acid or RNA) are found in nature and mediate phosphodiester bond cleavage and formation and peptide bond formation. Artificial ribozymes have been obtained by means of combinatorial chemistry approaches, such as in vitro selection and in vitro evolution [1], and have been shown to catalyze quite a broad array of other chemical reactions [2,3]. Deoxyribozymes or DNAzymes (enzymes made of DNA) are artificial molecules and are not found in nature. Although nucleic acids enzymes are still considered to act slowly compared with their proteinaceous counterparts, they are often a lot smaller, readily available and easier to study so that many details concerning their catalytic and molecular recognition mechanisms can be unravelled. Although the discovery of natural ribozymes dates back more than two decades, questions like How do natural ribozymes achieve catalysis? and To what extent can their catalytic mechanisms be compared with those of protein enzymes? still burn in the scientific community. The vast body of research in this field has been recently extensively reviewed [2,4 8] and will not be further considered here. Moreover, besides the pure scientific interest, it should not be forgotten that nucleic acid enzymes are currently and actively studied as potential molecular therapeutics. These studies are, at least in some cases, at such an advanced stage that phase I and II clinical trials are underway [9 11]. This article aims to highlight developments in the field of artificial nucleic acid enzymes in the past two years. New catalytic activities have been discovered for both ribozymes and DNAzymes. Several studies have expanded the scope and applicability of previously selected nucleic acid enzymes or have tried to elucidate the mechanism used to support catalytic activity. Allosterically regulated ribozymes will also briefly be considered; these artificial systems actually predate the discovery of natural riboswitches, with catalytic activity possibly modulated through metabolite RNA binding. Non-natural ribozymes Despite the lack of chemical diversity characterizing the array of functional groups present in RNA, relative to proteins, ribozymes with unprecedented catalytic activities are continuously being discovered by means of in 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 development of novel biotechnological processes. For example, nucleic acid catalysts developed for a practically relevant organic transformation could be immobilized on solid supports [13], in analogy to current technologies for immobilized enzymes [14]. Ribozymes showing redox activity have been developed in Suga s laboratory [15,16]. An alcohol dehydrogenase ribozyme was selected in the presence of NAD + and Zn 2+ and was found to oxidize a tethered benzyl alcohol substrate to the corresponding aldehyde in a strict cofactor-dependent fashion [15 ]. Additionally, one representative clone obtained from this in vitro selection was later found to catalyze the reverse reaction as well [16]. The appended benzaldehyde derivative could be reduced to the corresponding alcohol in the presence of NADH and Zn 2+, demonstrating for the first time that ribozymes can sustain reversible redox chemistry. Eaton s group [17 ] has reported the selection of a ribozyme that promotes the formation of a urea bond between peptide phosphonate substrates and the exocyclic amino group of the 3 0 -terminal cytidine residue of the ribozyme. These particular substrates were employed with the aim www.sciencedirect.com

Nucleic acid enzymes Fiammengo and Jäschke 615 of directly influencing the ribozyme s molecular recognition ability for substrates with differences at a distal site (away from the actual reactive group). An unusual selection strategy was therefore designed to isolate the active nucleic acid sequences, that is those catalyzing conjugation of the substrate to RNA via urea bond formation. The peptide RNA conjugates were captured with human neutrophile elastase, taking advantage of the known activity of peptide phosphonate as a suicide inhibitor for this enzyme (Figure 1). The selected catalysts mediate urea bond formation at the N terminus of the peptides and differentiate between substrates with the opposite configuration to the C-terminal residue. Ribozymes able to synthesize purine nucleotides have been selected [18 ]. Together with the already known ability of RNA to catalyze the synthesis of pyrimidine nucleotides [19], the results reported by the Unrau group [18 ] show that RNA is able to synthesize all the building blocks from which it is constituted. The same group has also reported two methodological studies aimed at solving the problem of identifying a ribozyme s core motif [20,21 ]. Extraneous sequences found in loops or beyond the 5 0 and the 3 0 boundaries of a ribozyme and unnecessary for catalytic activity are easily recognizable and removable. By contrast, it may prove extremely difficult to shorten interhelical joining regions by rational design, even when these sequences are poorly conserved, indicating a secondary role in catalysis. Each of the two reported strategies was applied to one of the three 4S U synthase ribozyme families previously identified [19]. Characterization of the core motif of family A was achieved by the construction of large libraries of deletion and mutation variants with as little sequence bias as possible [20]. The best way to achieve balanced levels of deletion proved to be a partial reblocking/deblocking strategy. After seven rounds of selection aimed at the isolation of short functional ribozymes, the mean pool length was decreased from 163 to 131 nucleotides with a net deletion frequency within the variablelength regions of 41%. The second strategy was applied to family B of the 4S U synthase ribozyme and is based on nonhomologous or random recombination [21 ]. Double-stranded DNA corresponding to the sequence of a previously isolated ribozyme was partially digested with DNase I, and sticky ends were filled using T4 DNA polymerase. The bluntend fragments were then reassembled into new molecules that had a broad sequence length distribution by reaction with T4 DNA ligase. PCR allowed selection and amplification of all molecules that had the 5 0 - and 3 0 - primer sequences at the corresponding end (10 8 DNA sequences), irrespective of internal deletions, inversions and translocations. After size-dependent in vitro selection, the original 271-nucleotide-long ribozyme was reduced to sequences as short as 81 nucleotides. RNA is not only able to synthesize its building blocks, but can also catalyze a templated primer extension reaction analogously to polymerase enzymes [22]. A novel strategy was developed to measure the processivity of a polymerase ribozyme showing that despite its inefficiency the ribozyme is undoubtedly partially processive [23]. Joyce and coworkers [24 ] showed that a self-replicating ribozyme could be converted to a cross-catalytic replication system in which two ribozymes catalyze each other s synthesis from four component substrates [24 ] (Figure 2). Two papers were concerned with ribozymes catalyzing aminoacylation of RNA substrates [25,26]. This function 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

616 Chemical biotechnology Figure 2 Cross-catalytic replication of a ligase ribozyme. Ribozymes T and T 0 selectively catalyze the ligation of substrates A 0 with B 0 and A with B, respectively. Dissociation of the product complex TT 0 generates new free copies of the two ribozymes leading to an autocatalytic behaviour of the system. enzymes but, given the fact that protein synthesis in the ribosome is actually carried out by RNA [27,28], it seems reasonable to suppose that in an ancestral world the synthetases could have also been RNA catalysts. A 45- nucleotide-long trna aminoacylation ribozyme was selected evolving a previously identified sequence [25]. This catalyst showed improved catalytic activity and is able to aminoacylate several trna in trans (not as a selfmodifier) with phenylalanine derivatives, provided that the correct three-nucleotide sequence is present at the 3 0 end of the trna. The second aminoacylation catalyst reported had the peculiarity of using coenzyme A (CoA) thioesters as reactants for the aminoacylation reaction [26]. Here, the ribozyme acted in cis (as a self-modifier), catalyzing the aminoacylation of the 2 0 -hydroxyl group of a specific uridine residue. Although much information is available on the structural characterization of natural RNA catalysts and the mechanisms involved in phosphodiester transfer, very little is known about ribozymes that catalyze other reactions. Two recent papers from the Jäschke laboratory [29,30 ] described the structural characterization of a small ribozyme catalyzing the Diels Alder reaction between anthracene and maleimide derivatives. Extensive mutation analysis and chemical and enzymatic probing experiments were used to identify and clarify the role of secondary and tertiary interactions in catalysis [30 ]. Unexpectedly, the data indicated the existence of a preformed catalytic pocket (i.e. no major rearrangement in the RNA structure upon substrate binding). These findings were fully supported by inspection of the ribozyme crystal structure obtained both in the absence of substrate and in the presence of tethered Diels Alder product [29 ]. The structural characterization of the active site suggests that catalysis is achieved via an almost perfect shape complementarity with the transition state, in combination with electronic contributions such as stacking of the anthracene substrate with adenine A3 and uridine U45 and hydrogen bonding to one carbonyl oxygen of the maleimide (Figure 3). Finally, no evidence for the involvement of metal ions in catalysis was found. By contrast, the Diels Alderase ribozyme isolated by the Eaton group required the presence of Cu 2+ and now, in a mutated sequence, of Cu 2+ and Ni 2+ [31]. The DNA pool for the new reported selection was generated by chemical synthesis mutation of a previously selected Diels Alderase ribozyme sequence at a rate of 25% per nucleotide. After 11 cycles of selection the obtained isolates showed, in all cases, substantially improved substrate binding (lower K m ) compared with the original sequence. The absolute need for metal ions suggests the occurrence of Lewis acid catalysis that has been stimulated during www.sciencedirect.com

Nucleic acid enzymes Fiammengo and Jäschke 617 Figure 3 The catalytic active site of the Diels Alder ribozyme. The structure reveals very good shape complementarity with the bound product (shown in blue). No metal ion is involved in interactions with the product. Hydrogen bonds are shown as dotted lines. The guanosine shown in green is the only unpaired nucleotide in the catalytic center. selection by using pyridyl-appended uridine derivatives in place of uridine. The use of such modified nucleotides has also allowed the isolation of RNA sequences able to induce the formation of palladium nanoparticles [32]. Although these sequences cannot be (and were not) defined as ribozymes, they promote the formation of metal metal bonds affording thin hexagonal palladium particles (1.3 0.6 mm, thickness 20 nm) in reaction times as short as 1 min. By comparison the initial random pool produced, in two hours, small particles of undefined shape with 5 nm diameter. This unprecedented activity of RNA suggests that perhaps even nowadays RNA can actively take part in the evolution of inorganic materials. DNAzymes DNAzymes have so far never been observed in nature and are therefore exclusively synthetic entities isolated through in vitro selection and evolution strategies. A review dealing with the recent developments in this field has been published in September 2004 [33]. Silverman and coworkers have isolated a multitude of DNAzymes that catalyze RNA ligation [33]. Catalysts with different properties have been obtained depending on the selection format. The first example of a DNAzyme catalyzing the formation of linear 3 0 5 0 linkages between two RNA substrates was obtained by rational design of the selection strategy [34], using many results from previous selections. To favor the formation of linear versus branched linkages between the 3 0 -OH of one RNA strand and the 5 0 -triphosphate of the other, the nascent ligation junction was embedded within a duplex (DNA:RNA) region. These catalysts are relatively slow (k obs = 0.008 min 1 for the most active clone) but specifically produce natural 3 0 5 0 junctions at 40 mm Mg 2+ and ph 9.0 [34]. Remarkably, no 2 0 5 0 junctions were observed. Unfortunately, sequence generality was seriously hampered by the minimal requirement of five specific RNA nucleotides around the ligation site and, for optimal activity, as many as eight RNA nucleotides had to be conserved. The development of 3 0 5 0 RNA ligase DNAzymes with broad generality for RNA substrates still remains a burning issue. Further selection attempts were undertaken by moving the ligation site from the duplex region to a short overhang so that no bias toward any specific junction mode was implemented in the selection [35 ]. Two different RNA substrates were used in two different selections. The results showed that different substrates produced very different selection outcomes. It was therefore concluded that the ligation site and the junction type (linear versus branched and 3 0 5 0 or 2 0 5 0 ; Figure 4) strongly depend on the substrate employed during the selection. This would be a very unfortunate occurrence in the search for generally applicable 3 0 5 0 ligase DNAzymes. It was actually possible, however, to shift the selection outcome towards the desired 3 0 5 0 linkage by implementing a cleavage step with an 8 17 DNAzyme [36,37]. In this way, all sequences catalyzing the correct 3 0 5 0 RNA products could be selectively cleaved, identified, and separated using gel electrophoresis during successive selection cycles [35 ]. The isolation of an RNA ligase DNAzyme requiring Zn 2+ for catalytic activity has also been reported [38]. In the presence of 1 mm Zn 2+ these catalysts promote the formation of a variety of RNA linkages: linear 3 0 5 0 (not obtained in the analogous selection in the presence of Mg 2+ ), 2 0 5 0,3 0 2 0,2 0 2 0, and branched 2 0 2 0 (3 0 ). RNA-cleaving DNAzymes have been extensively studied and proposed as valuable tools (e.g. as sensors for metal ions [39,40]) or for the creation of nanoscale mobile devices [41]. They also have potential use in functional genomics and gene therapy [10]. DNAzymes of the 8 17 family are versatile RNA-cleaving catalysts with k obs as high as 0.01 min 1 observed in the presence of divalent metal ions. It has been shown that 8 17 DNAzymes have the ability to cleave 14 of the 16 possible dinucleotide junctions [36]. Attempts to control the reactivity of 8 17 DNAzymes by attachment of photoresponsive groups have also been reported [42,43]. Schlosser and Li [44] have analyzed in detail how sequence diversity is affected by stringency (in the present case induced by shortening of the reaction time) during in vitro selection for RNA-cleaving DNAzymes. A logarithmic decrease in sequence diversity was observed with decrease in reaction time. www.sciencedirect.com

618 Chemical biotechnology Figure 4 Mode of action of RNA ligase DNAzymes with a ligation site on a short overhang (UAXCX). (a) DNAzyme substrate construct. The DNAzyme is in blue and the RNA substrate in red. Green arrows show the possible formation of 2 0 5 0 branched junctions. The black arrow shows the possible formation of 2 0 5 0 or 3 0 5 0 linear junctions. (b) Linear junction formation. (c) Branched junction formation. DNAzymes able to catalyze RNA hydrolysis in the absence of divalent metal ions have been obtained by expanding the array of chemical functionality of DNA. Modified bases carrying imidazole and alkyl primary amino groups have been used to this end [45,46]. In one case, multiple turnover was obtained for the first time in a trans assay employing a DNA RNA chimeric substrate [45]. DNAzymes with sequences carrying the two above mentioned additional functional groups were also selected for cleavage of DNA substrates at abasic sites, therefore displaying apurinic/apyrimidinic lyase endonuclease activity [47 ]. Allosteric ribozymes and riboswitches with catalytic activity The catalytic activity of allosterically regulated ribozymes is modulated by the binding of a suitable effector. In vitro selection strategies for allosteric ribozymes (also named aptazymes) generally start from a pre-existing ribozyme domain to which a randomized RNA domain is appended. An allosteric selection procedure is then carried out to select sequences that show catalytic acivity only if binding of the effector to the RNA occurs. The classical way to implement an allosteric selection therefore requires an appropriate counter selection step to remove all sequences active in the absence of the effector. Using this activity-based selection strategy, however, it was not possible to select for aspartame-dependent hammerhead ribozymes [48]. A new hybrid strategy for allosteric ribozyme evolution has been proposed, which allows the preparation of RiboReporter TM sensors for aspartame and caffeine [48]. Hammerhead-ribozyme-based pools were first enriched for sequences binding aspartame or caffeine using a standard SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure developed for the selection of aptamers [49]. Subsequently, several rounds of activity-based selection were performed leading to the isolation of catalytically active sequences responsive to the presence of the desired effector. Interestingly, this procedure led to the successful isolation of the desired aspartame-dependent ribozyme only after introduction of mutagenesis between the binding-based and the actvity-based selection steps. Another allosteric selection starting with a hammerheadribozyme-based pool had the goal of developing metal- www.sciencedirect.com

Nucleic acid enzymes Fiammengo and Jäschke 619 binding ribozymes [50]. The positive selection was carried out in the presence of a cocktail of metal ions: Mg 2+, K +,Li +,Na +,Rb +,Ca 2+,Sr 2+,Cd 2+,Co 2+,Mn 2+,Ni 2+ and Zn 2+, whereas only Mg 2+ was present during the counterselection. Five different classes of ribozymes were isolated from this selection with significantly increased activity in the presence of Cd 2+,Co 2+,Mn 2+,Ni 2+ and Zn 2+. None of the selected ribozymes was found to respond to Ca 2+,Sr 2+ or any of the monovalent cations. Discrimination among the five identified effectors was not observed, however. Ideal allosteric ribozymes should possess high activation factors, defined as the ratio between the rate of the catalyzed reaction in the presence and in the absence of the effector. Complete inactivity should be observed in the absence of the effector. Towards this goal, stringent counterselection procedures are generally performed. Nevertheless, two examples have recently shown that it may prove very difficult to reduce the catalytic activity of the starting pool to a background level. The problem was encountered during the selection of a peptide-dependent ribozyme ligase [51] as well as during the selection of the first allosteric ribozyme catalyzing the reaction between two small non-rna substrates, a theophylline-dependent Diels Alderase ribozyme [52]. Elaborating on the idea of allosteric ribozymes, RNA catalysts with endonuclease type activity controlled by a novel specific on/off adaptor (SOFA-ribozymes) have been reported. In brief, these ribozymes are locked in an inactive state in the absence of the target substrate. One part of the structure is acting as biosensor and in the presence of the substrate the catalytic activity is switched on leading to the cleavage of the substrate [53]. These molecules, derived from rational design engineering of the hepatitis delta virus ribozyme, are in their mode of action on the borderline between allosteric ribozymes and riboswitches. Riboswitches are highly conserved domains found in mrna that can regulate gene expression by sensing the concentration of relevant metabolites through their direct binding [54,55 ]. In 2003, Breaker s group [56] first proposed that gene regulation in response to binding of thiamine pyrophosphate to the competent riboswitches in plants and in other eukaryotes might involve direct mrna processing or RNA splicing. This observation implied that those riboswitches would possibly behave as allosterically regulated ribozymes. One year later the same group reported the first example of a riboswitch working as a metabolite-responsive ribozyme discovered in Gram-positive bacteria [55 ]. This ribozyme is activated by glucosamine-6-phosphate (GlcN6P) and cleaves the mrna of the glms gene in response to an increasing concentration of GlcN6P, which is the product of the enzyme encoded by the glms gene itself [57]. At present, however, it is not possible to determine whether this riboswitch functions as a true allosteric ribozyme or if GlcN6P is taking an active part to the catalytic process. Conclusions Novel catalytic activities have enriched the arsenal of reactions catalyzed by nucleic acid enzymes, thus showing once again the versatility of this class of biopolymers. In most cases nucleic acid catalysts are less efficient than their proteinaceous counterparts. Even nature, however, during the long history of evolution has selected (or perhaps conserved) RNA with catalytic activity for specific and important functions such as protein synthesis in the ribosome. Maybe this relative inefficiency is mostly caused by our unrefined and limited way of selecting nucleic acid enzymes. It is clear that the successful isolation of nucleic acid enzymes from large combinatorial libraries strictly depends on the further development of our present in vitro selection and evolution techniques. Several studies are beginning to unravel the mysteries behind this collection of intricate and ingenious procedures. Meanwhile, original solutions are requested almost every time that a new selection procedure is developed. Nevertheless, even with the current limitations, this field is quickly progressing. We assume that further advances will be made towards the application of some of the already selected nucleic acid enzymes in various fields ranging from molecular medicine to the development of new sensors. We also forecast that other catalytic activities will be found, possibly allowing the development of nucleic acid enzymes as on-demand tools for solving highly relevant chemical transformations with specificity and stereoselectivity. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Joyce GF: Directed evolution of nucleic acid enzymes. Annu Rev Biochem 2004, 73:791-836. 2. Lilley DM: Structure, folding and mechanisms of ribozymes. Curr Opin Struct Biol 2005, 15:313-323. 3. Jäschke A, Seelig B: Evolution of DNA and RNA as catalysts for chemical reactions. Curr Opin Chem Biol 2000, 4:257-262. 4. Woodson SA: Structure and assembly of group I introns. Curr Opin Struct Biol 2005, 15:324-330. 5. Fedor MJ, Williamson JR: The catalytic diversity of RNAs. Nat Rev Mol Cell Biol 2005, 6:399-412. 6. Doudna JA, Lorsch JR: Ribozyme catalysis: not different, just worse. Nat Struct Mol Biol 2005, 12:395-402. 7. Emilsson GM, Nakamura S, Roth A, Breaker RR: Ribozyme speed limits. RNA 2003, 9:907-918. 8. Breaker RR, Emilsson GM, Lazarev D, Nakamura S, Puskarz IJ, Roth A, Sudarsan N: A common speed limit for RNA-cleaving ribozymes and deoxyribozymes. RNA 2003, 9:949-957. www.sciencedirect.com

620 Chemical biotechnology 9. Bagheri S, Kashani-Sabet M: Ribozymes in the age of molecular therapeutics. Curr Mol Med 2004, 4:489-506. 10. Dass CR: Deoxyribozymes: cleaving a path to clinical trials. Trends Pharmacol Sci 2004, 25:395-397. 11. Peracchi A: Prospects for antiviral ribozymes and deoxyribozymes. Rev Med Virol 2004, 14:47-64. 12. Joyce GF: Molecular evolution: booting up life. Nature 2002, 420:278-279. 13. Schlatterer JC, Stuhlmann F, Jäschke A: Stereoselective synthesis using immobilized Diels-Alderase ribozymes. ChemBioChem 2003, 4:1089-1092. 14. Cao L, van Langen L, Sheldon RA: Immobilized enzymes: carrier-bound or carrier-free? Curr Opin Biotechnol 2003, 14:387-394. 15. Tsukiji S, Pattnaik SB, Suga H: An alcohol dehydrogenase ribozyme. Nat Struct Biol 2003, 10:713-717. The selected ribozymes achieve rate enhancement of 10 7. A minimal catalytic motif only 76 nucleotide long and having identical activity as the wild-type sequence, could be identified. 16. Tsukiji S, Pattnaik SB, Suga H: Reduction of an aldehyde by a NADH/Zn 2+ -dependent redox active ribozyme. J Am Chem Soc 2004, 126:5044-5045. 17. Nieuwlandt D, West M, Cheng X, Kirshenheuter G, Eaton BE: The first example of an RNA urea synthase: selection through the enzyme active site of human neutrophile elastase. ChemBioChem 2003, 4:651-654. Unexpectedly, urea bond formation did not take place at the expected primary amino group tethered to the RNA sequences via a flexible polyethylene glycol spacer. 18. Lau MW, Cadieux KE, Unrau PJ: Isolation of fast purine nucleotide synthase ribozymes. J Am Chem Soc 2004, 126:15686-15693. These ribozymes catalyze the reaction between 6-thioguanine and 5- phosphoribosyl-1-pyrophosphate to give 6-thioguanine monophosphate with apparent efficiency (k cat app /K m ) as high as 284 M 1 min 1. 19. Unrau PJ, Bartel DP: RNA-catalyzed nucleotide synthesis. Nature 1998, 395:260-263. 20. Chapple KE, Bartel DP, Unrau PJ: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme. RNA 2003, 9:1208-1220. 21. Wang QS, Unrau PJ: Ribozyme motif structure mapped using random recombination and selection. RNA 2005, 11:404-411. This experimental method seems a very efficient way of determining a ribozyme core motif in an unbiased way and it is very likely that it will have a great impact in future research on catalytically active RNA structures. 22. Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP: RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science 2001, 292:1319-1325. 23. Lawrence MS, Bartel DP: Processivity of ribozyme-catalyzed RNA polymerization. Biochemistry 2003, 42:8748-8755. 24. Kim DE, Joyce GF: Cross-catalytic replication of an RNA ligase ribozyme. Chem Biol 2004, 11:1505-1512. The rate of cross-catalytic replication observed for this system was only modest, but the efficiency and specificity of the two-component reactions were sufficient to ensure that the cross-catalytic pathway was dominating over the possible formation of promiscuous products. 25. Murakami H, Saito H, Suga H: A versatile trna aminoacylation catalyst based on RNA. Chem Biol 2003, 10:655-662. 26. Li N, Huang F: Ribozyme-catalyzed aminoacylation from CoA thioesters. Biochemistry 2005, 44:4582-4590. 27. Baram D, Yonath A: From peptide-bond formation to cotranslational folding: dynamic, regulatory and evolutionary aspects. FEBS Lett 2005, 579:948-954. 28. Weinger JS, Parnell KM, Dorner S, Green R, Strobel SA: Substrate-assisted catalysis of peptide bond formation by the ribosome. Nat Struct Mol Biol 2004, 11:1101-1106. 29. Serganov A, Keiper S, Malinina L, Tereshko V, Skripkin E, Höbartner C, Polonskaia A, Phan AT, Wombacher R, Micura R et al.: Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation. Nat Struct Mol Biol 2005, 12:218-224. The catalytic site architecture reveals that catalysis is achieved by a combination of proximity, complementarity, and electronic effects. Striking structural parallels are observed with the active site of a catalytic antibody evolved for a similar reaction and involving anthracene as the diene. This suggests that common structural principles and catalytic mechanisms are at work in macromolecule-mediated catalysis of Diels Alder reactions. 30. Keiper S, Bebenroth D, Seelig B, Westhof E, Jäschke A: Architecture of a Diels-Alderase ribozyme with a preformed catalytic pocket. Chem Biol 2004, 11:1217-1227. Unlike many other biopolymeric systems, this ribozyme does not show induced fit recognition upon substrate binding. 31. Tarasow TM, Kellogg E, Holley BL, Nieuwlandt D, Tarasow SL, Eaton BE: The effect of mutation on RNA Diels-Alderases. J Am Chem Soc 2004, 126:11843-11851. 32. Gugliotti LA, Feldheim DL, Eaton BE: RNA-mediated metal-metal bond formation in the synthesis of hexagonal palladium nanoparticles. Science 2004, 304:850-852. 33. Silverman SK: Deoxyribozymes: DNA catalysts for bioorganic chemistry. Org Biomol Chem 2004, 2:2701-2706. 34. Coppins RL, Silverman SK: Rational modification of a selection strategy leads to deoxyribozymes that create native 3 0-5 0 RNA linkages. J Am Chem Soc 2004, 126:16426-16432. 35. Wang Y, Silverman SK: Directing the outcome of deoxyribozyme selections to favor native 3 0-5 0 RNA ligation. Biochemistry 2005, 44:3017-3023. The substrates used in these selections were related either to the natural sequences of spliceosome substrates or to group II introns. Even an already evolved population of DNAzymes, estimated to contain less than 1% sequences catalyzing 3 0 5 0 linkages, gave, after only four additional selection cycles, 23 out of 24 isolated clones with the desired 3 0 5 0 ligase activity. 36. Cruz RPG, Withers JB, Li Y: Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme. Chem Biol 2004, 11:57-67. 37. Santoro SW, Joyce GF: A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 1997, 94:4262-4266. 38. Hoadley KA, Purtha WE, Wolf AC, Flynn-Charlebois A, Silverman SK: Zn 2+ -Dependent deoxyribozymes that form natural and unnatural RNA linkages. Biochemistry 2005, 44:9217-9231. 39. Liu J, Lu Y: A colorimetric lead biosensor using DNAzymedirected assembly of gold nanoparticles. J Am Chem Soc 2003, 125:6642-6643. The activity of an 8 17 DNAzyme mediates the degree of aggregation of DNA-passivated gold nanoparticles (resulting in different colors), depending on the concentration of Pb 2+. 40. Thomas JM, Ting R, Perrin DM: High affinity DNAzyme-based ligands for transition metal cations a prototype sensor for Hg 2+. Org Biomol Chem 2004, 2:307-312. 41. Chen Y, Wang M, Mao C: An autonomous DNA nanomotor powered by a DNA enzyme. Angew Chem Int Ed Engl 2004, 43:3554-3557. 42. Ting R, Lermer L, Perrin DM: Triggering DNAzymes with light: a photoactive C8 thioether-linked adenosine. J Am Chem Soc 2004, 126:12720-12721. 43. Liu Y, Sen D: Light-regulated catalysis by an RNA-cleaving deoxyribozyme. J Mol Biol 2004, 341:887-892. 44. Schlosser K, Li Y: Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection. Biochemistry 2004, 43:9695-9707. 45. Ting R, Thomas JM, Lermer L, Perrin DM: Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes www.sciencedirect.com

Nucleic acid enzymes Fiammengo and Jäschke 621 RNA hydrolysis independent of a divalent metal cation. Nucleic Acids Res 2004, 32:6660-6672. 46. Sidorov AV, Grasby JA, Williams DM: Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities. Nucleic Acids Res 2004, 32:1591-1601. 47. May JP, Ting R, Lermer L, Thomas JM, Roupioz Y, Perrin DM: Covalent Schiff base catalysis and turnover by a DNAzyme: A M 2+ -independent AP-endonuclease mimic. J Am Chem Soc 2004, 126:4145-4156. The formation of a Schiff base intermediate between the abasic DNA substrate and a primary amino group carried by the catalyst was inferred by several data including the actual identification of a product catalyst Schiff base adduct. 48. Ferguson A, Boomer RM, Kurz M, Keene SC, Diener JL, Keefe AD, Wilson C, Cload ST: A novel strategy for selection of allosteric ribozymes yields RiboReporter sensors for caffeine and aspartame. Nucleic Acids Res 2004, 32:1756-1766. 49. Famulok M, Mayer G, Blind M: Nucleic acid aptamers from selection in vitro to applications in vivo. Acc Chem Res 2000, 33:591-599. 50. Zivarts M, Liu Y, Breaker RR: Engineered allosteric ribozymes that respond to specific divalent metal ions. Nucleic Acids Res 2005, 33:622-631. 51. Robertson MP, Knudsen SM, Ellington AD: In vitro selection of ribozymes dependent on peptides for activity. RNA 2004, 10:114-127. 52. Helm M, Petermeier M, Ge B, Fiammengo R, Jäschke A: Allosterically activated Diels-Alder catalysis by a ribozyme. J Am Chem Soc 2005, 127:10492-10493. 53. Bergeron LJ, Perreault J-P: Target-dependent on/off switch increases ribozyme fidelity. Nucleic Acids Res 2005, 33:1240-1248. 54. Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS: Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet 2004, 20:44-50. 55. Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR: Control of gene expression by a natural metabolite-responsive ribozyme. Nature 2004, 428:281-286. By correlating the in vitro ribozyme activity with the in vivo activity of a b- galactosidase reporter gene, it was possible to show that the ribozyme activity represses the glms gene, thus keeping the production of GlcN6P under control. 56. Sudarsan N, Barrick JE, Breaker RR: Metabolite-binding RNA domains are present in the genes of eukaryotes. RNA 2003, 9:644-647. 57. Milewski S: Glucosamine-6-phosphate synthase the multi-facets enzyme. Biochim Biophys Acta 2002, 1597:173-192. www.sciencedirect.com