Nucleic Roberto Fiammengo and Andres Ja¨ schke

Since the discovery of the first natural more than 20 extensively reviewed [2,4–8] and will not be further years ago, it has become clear that nucleic 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]. , 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 , 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 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 ’ 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 cleavage and immobilized enzymes [14]. formation and 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 enzymes?’ still burn in the scientific . group of the 30-terminal residue of the ribozyme. The vast body of research in this field has been recently These particular substrates were employed with the aim

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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 . 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 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 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--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 - 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 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 leading to an autocatalytic behaviour of the system. enzymes but, given the fact that protein synthesis in the of secondary and tertiary interactions in catalysis [30]. ribosome is actually carried out by RNA [27,28], it seems Unexpectedly, the data indicated the existence of a reasonable to suppose that in an ancestral world the preformed catalytic pocket (i.e. no major rearrangement synthetases could have also been RNA catalysts. A 45- in the RNA structure upon substrate binding). These nucleotide-long tRNA aminoacylation ribozyme was findings were fully supported by inspection of the ribo- selected evolving a previously identified sequence [25]. zyme crystal structure obtained both in the absence of This catalyst showed improved catalytic activity and is substrate and in the presence of tethered Diels–Alder able to aminoacylate several tRNA in trans (not as a self- product [29]. The structural characterization of the modifier) with phenylalanine derivatives, provided that active site suggests that catalysis is achieved via an almost the correct three-nucleotide sequence is present at the 30 perfect shape complementarity with the transition state, end of the tRNA. The second aminoacylation catalyst in combination with electronic contributions such as reported had the peculiarity of using coenzyme A (CoA) stacking of the anthracene substrate with A3 thioesters as reactants for the aminoacylation reaction and U45 and hydrogen bonding to one carbonyl [26]. Here, the ribozyme acted in cis (as a self-modifier), of the maleimide (Figure 3). Finally, no evidence catalyzing the aminoacylation of the 20-hydroxyl group of for the involvement of metal ions in catalysis was found. a specific uridine residue. By contrast, the Diels–Alderase ribozyme isolated by the Although much information is available on the structural Eaton group required the presence of Cu2+ and now, in a characterization of natural RNA catalysts and the mutated sequence, of Cu2+ and Ni2+ [31]. The DNA pool mechanisms involved in phosphodiester transfer, very for the new reported selection was generated by chemical little is known about ribozymes that catalyze other reac- synthesis mutation of a previously selected Diels–Alder- tions. Two recent papers from the Ja¨schke laboratory ase ribozyme sequence at a rate of 25% per nucleotide. [29,30] described the structural characterization of a After 11 cycles of selection the obtained isolates showed, small ribozyme catalyzing the Diels–Alder reaction in all cases, substantially improved substrate binding between anthracene and maleimide derivatives. Exten- (lower Km) compared with the original sequence. The sive mutation analysis and chemical and enzymatic prob- absolute need for metal ions suggests the occurrence of ing experiments were used to identify and clarify the role Lewis acid catalysis that has been stimulated during

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Figure 3 1 (kobs = 0.008 min for the most active clone) but speci- fically produce ‘natural’ 30–50 junctions at 40 mM Mg2+ and pH 9.0 [34]. Remarkably, no 20–50 junctions were observed. Unfortunately, sequence generality was ser- iously 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 30–50 RNA ligase DNAzymes with broad generality for RNA substrates still remains a burn- ing 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 The catalytic active site of the Diels–Alder ribozyme. The structure reveals very good shape complementarity with the bound product produced very different selection outcomes. It was there- (shown in blue). No metal ion is involved in interactions with the product. fore concluded that the ligation site and the junction type Hydrogen bonds are shown as dotted lines. The shown in (linear versus branched and 30–50 or 20–50; Figure 4) green is the only unpaired nucleotide in the catalytic center. strongly depend on the substrate employed during the selection. This would be a very unfortunate occurrence in selection by using pyridyl-appended uridine derivatives the search for generally applicable 30–50 ligase DNA- in place of uridine. zymes. It was actually possible, however, to shift the selection outcome towards the desired 30–50 linkage by The use of such modified nucleotides has also allowed the implementing a cleavage step with an 8–17 DNAzyme isolation of RNA sequences able to induce the formation [36,37]. In this way, all sequences catalyzing the correct of palladium nanoparticles [32]. Although these 30–50 RNA products could be selectively cleaved, identi- sequences cannot be (and were not) defined as ribozymes, fied, and separated using during suc- they promote the formation of metal–metal bonds afford- cessive selection cycles [35]. ing thin hexagonal palladium particles (1.3 0.6 mm, thickness 20 nm) in reaction times as short as 1 min. The isolation of an RNA ligase DNAzyme requiring Zn2+ By comparison the initial random pool produced, in two for catalytic activity has also been reported [38]. In the hours, small particles of undefined shape with 5nm presence of 1 mM Zn2+ these catalysts promote the for- diameter. This unprecedented activity of RNA suggests mation of a variety of RNA linkages: linear 30–50 (not that perhaps even nowadays RNA can actively take part obtained in the analogous selection in the presence of in the evolution of inorganic materials. Mg2+), 20–50,30–20,20–20, and branched 20–20 (30).

DNAzymes RNA-cleaving DNAzymes have been extensively stu- DNAzymes have so far never been observed in nature and died and proposed as valuable tools (e.g. as sensors for are therefore exclusively synthetic entities isolated metal ions [39,40]) or for the creation of nanoscale mobile through in vitro selection and evolution strategies. A devices [41]. They also have potential use in functional review dealing with the recent developments in this field and therapy [10]. has been published in September 2004 [33]. DNAzymes of the ‘8–17’ family are versatile RNA-cleav- 1 Silverman and coworkers have isolated a multitude of ing catalysts with kobs as high as 0.01 min observed in DNAzymes that catalyze RNA ligation [33]. Catalysts the presence of divalent metal ions. It has been shown with different properties have been obtained depending that 8–17 DNAzymes have the ability to cleave 14 of the on the selection format. The first example of a DNAzyme 16 possible dinucleotide junctions [36]. Attempts to con- catalyzing the formation of linear 30–50 linkages between trol the reactivity of 8–17 DNAzymes by attachment of two RNA substrates was obtained by rational design photoresponsive groups have also been reported [42,43]. of the selection strategy [34], using many results from Schlosser and Li [44] have analyzed in detail how previous selections. To favor the formation of linear sequence diversity is affected by stringency (in the pre- versus branched linkages between the 30-OH of one sent case induced by shortening of the reaction time) RNA strand and the 50-triphosphate of the other, the during in vitro selection for RNA-cleaving DNAzymes. A nascent ligation junction was embedded within a duplex logarithmic decrease in sequence diversity was observed (DNA:RNA) region. These catalysts are relatively slow with decrease in reaction time. www.sciencedirect.com Current Opinion in Biotechnology 2005, 16:614–621 618 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 20–50 branched junctions. The black arrow shows the possible formation of 20–50 or 30–50 linear junctions. (b) Linear junction formation. (c) Branched junction formation.

DNAzymes able to catalyze RNA hydrolysis in the implement an allosteric selection therefore requires an absence of divalent metal ions have been obtained by appropriate counter selection step to remove all expanding the array of chemical functionality of DNA. sequences active in the absence of the effector. Using Modified bases carrying imidazole and alkyl primary this activity-based selection strategy, however, it was not amino groups have been used to this end [45,46]. In possible to select for aspartame-dependent hammerhead one case, multiple turnover was obtained for the first ribozymes [48]. A new hybrid strategy for allosteric ribo- time in a trans assay employing a DNA–RNA chimeric zyme evolution has been proposed, which allows the substrate [45]. DNAzymes with sequences carrying the preparation of RiboReporterTM sensors for aspartame two above mentioned additional functional groups were and caffeine [48]. Hammerhead-ribozyme-based pools also selected for cleavage of DNA substrates at abasic were first enriched for sequences binding aspartame or sites, therefore displaying apurinic/apyrimidinic lyase– caffeine using a standard SELEX (Systematic Evolution endonuclease activity [47]. of Ligands by EXponential enrichment) procedure devel- oped for the selection of [49]. Subsequently, Allosteric ribozymes and riboswitches with several rounds of activity-based selection were performed catalytic activity leading to the isolation of catalytically active sequences The catalytic activity of allosterically regulated ribozymes responsive to the presence of the desired effector. Inter- is modulated by the binding of a suitable effector. In vitro estingly, this procedure led to the successful isolation of selection strategies for allosteric ribozymes (also named the desired aspartame-dependent ribozyme only after aptazymes) generally start from a pre-existing ribozyme introduction of mutagenesis between the binding-based domain to which a randomized RNA domain is appended. and the actvity-based selection steps. An allosteric selection procedure is then carried out to select sequences that show catalytic acivity only if bind- Another allosteric selection starting with a hammerhead- ing of the effector to the RNA occurs. The classical way to ribozyme-based pool had the goal of developing metal-

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binding ribozymes [50]. The positive selection was car- however, it is not possible to determine whether this ried out in the presence of a cocktail of metal ions: Mg2+, riboswitch functions as a true allosteric ribozyme or if K+,Li+,Na+,Rb+,Ca2+,Sr2+,Cd2+,Co2+,Mn2+,Ni2+ and GlcN6P is taking an active part to the catalytic process. Zn2+, whereas only Mg2+ was present during the counter- selection. Five different classes of ribozymes were iso- Conclusions lated from this selection with significantly increased Novel catalytic activities have enriched the arsenal of activity in the presence of Cd2+,Co2+,Mn2+,Ni2+ and reactions catalyzed by nucleic acid enzymes, thus show- Zn2+. None of the selected ribozymes was found to ing once again the versatility of this class of . respond to Ca2+,Sr2+ or any of the monovalent cations. In most cases nucleic acid catalysts are less efficient than Discrimination among the five identified effectors was their proteinaceous counterparts. Even nature, however, not observed, however. during the long history of evolution has selected (or perhaps conserved) RNA with catalytic activity for spe- Ideal allosteric ribozymes should possess high activation cific and important functions such as protein synthesis in factors, defined as the ratio between the rate of the the ribosome. Maybe this relative inefficiency is mostly catalyzed reaction in the presence and in the absence caused by our unrefined and limited way of selecting of the effector. Complete inactivity should be observed in nucleic acid enzymes. It is clear that the successful the absence of the effector. Towards this goal, stringent isolation of nucleic acid enzymes from large combinatorial counterselection procedures are generally performed. libraries strictly depends on the further development of Nevertheless, two examples have recently shown that our present in vitro selection and evolution techniques. it may prove very difficult to reduce the catalytic activity Several studies are beginning to unravel the mysteries of the starting pool to a background level. The problem behind this collection of intricate and ingenious proce- was encountered during the selection of a peptide-depen- dures. Meanwhile, original solutions are requested almost dent ribozyme ligase [51] as well as during the selection of every time that a new selection procedure is developed. the first allosteric ribozyme catalyzing the reaction between two small non-RNA substrates, a theophyl- Nevertheless, even with the current limitations, this field line-dependent Diels–Alderase ribozyme [52]. is quickly progressing. We assume that further advances will be made towards the application of some of the Elaborating on the idea of allosteric ribozymes, RNA already selected nucleic acid enzymes in various fields catalysts with endonuclease type activity controlled by ranging from molecular medicine to the development of a novel specific on/off adaptor (SOFA-ribozymes) have new sensors. We also forecast that other catalytic activ- been reported. In brief, these ribozymes are locked in an ities will be found, possibly allowing the development of inactive state in the absence of the target substrate. One nucleic acid enzymes as on-demand tools for solving part of the structure is acting as and in the highly relevant chemical transformations with specificity presence of the substrate the catalytic activity is switched and stereoselectivity. on leading to the cleavage of the substrate [53]. These molecules, derived from rational design engineering of References and recommended reading the hepatitis delta ribozyme, are in their mode of Papers of particular interest, published within the annual period of review, have been highlighted as: action on the borderline between allosteric ribozymes and riboswitches. of special interest of outstanding interest Riboswitches are highly conserved domains found in 1. Joyce GF: of nucleic acid enzymes. mRNA that can regulate gene expression by sensing Annu Rev Biochem 2004, 73:791-836. the concentration of relevant metabolites through their 2. Lilley DM: Structure, folding and mechanisms of ribozymes. direct binding [54,55 ]. In 2003, Breaker’s group [56] first Curr Opin Struct Biol 2005, 15:313-323. proposed that gene regulation in response to binding of 3. Ja¨ schke A, Seelig B: Evolution of DNA and RNA as catalysts thiamine pyrophosphate to the competent riboswitches in for chemical reactions. Curr Opin Chem Biol 2000, plants and in other might involve direct 4:257-262. mRNA processing or RNA splicing. This observation 4. Woodson SA: Structure and assembly of group I introns. implied that those riboswitches would possibly behave Curr Opin Struct Biol 2005, 15:324-330. as allosterically regulated ribozymes. One year later the 5. Fedor MJ, Williamson JR: The catalytic diversity of . same group reported the first example of a riboswitch Nat Rev Mol Biol 2005, 6:399-412. working as a metabolite-responsive ribozyme discovered 6. Doudna JA, Lorsch JR: Ribozyme catalysis: not different, just worse. Nat Struct Mol Biol 2005, 12:395-402. in Gram-positive [55]. This ribozyme is acti- vated by glucosamine-6- (GlcN6P) and cleaves 7. Emilsson GM, Nakamura S, Roth A, Breaker RR: Ribozyme speed limits. RNA 2003, 9:907-918. the mRNA of the glmS gene in response to an increasing 8. Breaker RR, Emilsson GM, Lazarev D, Nakamura S, Puskarz IJ, concentration of GlcN6P, which is the product of the Roth A, Sudarsan N: A common speed limit for RNA-cleaving enzyme encoded by the glmS gene itself [57]. At present, ribozymes and . RNA 2003, 9:949-957. www.sciencedirect.com Current Opinion in Biotechnology 2005, 16:614–621 620 Chemical biotechnology

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