Ribozyme-catalysed carbon-carbon bond formation

Ribozymes and RNA Catalysis

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Polacek N.,Gaynor, M., Yassin, A. and Mankin. A. S. (200 I ) Nature (London) 4 I I , 498-50 I Thompson, J., Kim, D. F., O'Connor, M., Lieberman, K. R., Bayfield, M. A,, Gregory, S. T., Green, R, Noller, H. F. and Dahlberg, A. E. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9002-9007

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Das. G. K.. Bhattacharyya. D. and Burma. D. P. (1999) J. Theor. Biol. 200, 193-205

Received 27 August 2002

Selectivity and Novel RNA-Catalysed Activity Ribozyme-catalysed carbon-carbon bond formation A. Jaschke',F. Stuhlmann, D. Bebenroth, S. Keiper and R. Wombacher Department of Biology, Chemistry & Pharmacy, Institute of Chemistry, Freie Universitat Berlin, Thielallee 63, D- I 4 I95 Berlin, Germany bonds both describe Diels-Alder reactions. A comparison of these two systems was recently published [l]; therefore this review will focus primarily on the Diels-Alderase ribozymes developed in our group.

Abstract Numerous RNA molecules with new catalytic properties have been isolated from synthetic combinatorial libraries. A broad range of chemical reactions is catalysed, and nucleic acids can accelerate bond formation between small organic substrates. This review focuses on carbon-carbon bond formation accelerated by in vitro selected ribozymes. Mechanistic investigations and structure-function relationships are discussed.

The Diels-Alder reaction T h e Diels-Alder reaction is one of the most important C-C bond-forming processes in preparative organic chemistry. This transformation belongs to the class of pericyclic reactions and is a [4n+ 2x1 cycloaddition, usually between an electron-rich 1,3-diene and an electron-deficient dienophile. T h e reaction creates two C-C bonds and up to four new stereocentres. In the course of the reaction, a six-membered carbocycle is formed. Because six 7c electrons are involved, it has been viewed as proceeding through an aromatic transition state, which is more energetically favoured than a non-concerted reaction path.

Introduction Over the past two decades, it has become evident that RNA can catalyse a broad range of chemical reactions. Nucleic acids can fold into complex three-dimensional structures, including binding sites and catalytic centres, and provide an environment in which various reactions can be facilitated. It has been demonstrated that nucleic acids are able to accelerate the formation, cleavage and rearrangement of various types of covalent bonds, although most of these reactions do not occur towards small substrate molecules free in solution. T h e formation of carbon-carbon bonds is central to biochemistry, organic synthesis and prebiotic chemistry. Accordingly, C-C bond formation has been studied extensively from various perspectives. Although there are various strategies available for C-C bond formation in biochemistry and synthetic organic chemistry, the only two examples of ribozymes forming C-C

Direct selection of Diels-Alderase ribozymes Artificial ribozymes can be selected from synthetic combinatorial RNA libraries, which can contain more than 10ls different sequences. These enormous complexities can only be handled because nucleic acid molecules are genetically encoded, i.e. they carry the information for their own replication. This property allows for an iterative screening over several rounds until active species dominate the enriched libraries. Although in vitro selection of aptamers against immobilized transition state analogues did not produce DielsAlderase ribozymes [2], active catalysts were obtained by direct selection [3,4]. In both cases, linker-coupled (or tethered) reactants were used. T h e potential diene was

Key words Diek-Alder reaction, in vitro selection. stereoselectivity, substrate binding Abbreviation used LUMO, lowest-unoccupied molecular ohital 'To whom correspondence should be addressed, at the present address Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-UniversitatHeidelberg, Im Neuenheimer Feld 364, 69 I20 Heidelberg, Germany (e-mail jaeschke@uni-hd de)

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0 2002 Biochemical Society

Biochemical Society Transactions (2002) Volume 30, part 6

imide about 18 500-fold (Figure 1B). T h e nucleotide sequences in the bulge region were found to be rather conserved, while the helical stems are variable. T h e motif could be rationally converted into bi- and tripartite, highly active variants by formal strand breakage in one or both of the two loop regions and re-assembly of ribozymes from the two or three corresponding RNA fragments

covalently tethered to the members of the RNA library by suitable chemo-enzymic methods, and the resulting library of RNA-tether-diene conjugates was then incubated with a biotinylated dienophile. RNA molecules that accelerated the reaction of the tethered diene were thereby tagged with the biotinyl residue and could be easily isolated and selectively amplified. Cyclic repetition allowed enrichment of the most active catalysts. T h e ribozymes isolated by the Eaton group [3] accelerated the reaction of a tethered aliphatic diene with biotinylated maleimide about 700-fold. No activity was observed, however, towards the two free (i.e. non-tethered) reactants, and therefore no multiple turnover was achieved.

~41.

True stereoselective catalysis of a bimolecular cycloaddition We subsequently demonstrated that the selected Diels-Alderase ribozymes are able to accelerate C-C bond formation in a true bimolecular fashion [S]. Organic molecules as small as 9-hydroxymethylanthracene and N-ethylmaleimide are specifically recognized by the 49-mer minimum ribozyme, followed by conversion into the respective Diels-Alder products and product dissociation from the catalyst (Figure 2). T h e ribozyme performs the reaction with multiple turnovers, and a k,,,, of approx. 20min-' was measured, placing this RNA among the faster ribozymes. Saturation-type kinetics with respect to both reactants, as well as product inhibition, was observed. In the aforementioned study [S], another characteristic feature of enzymic catalysis was demonstrated for ribozymes : enantioselective bond formation. Whereas the uncatalysed reaction produces racemic product mixtures, the ribozyme-catalysed conversion shows an enantioselectivity of over 95 enantiomeric excess. T h e

Diels-Alder reactions with aromatic dienes Our laboratory demonstrated that unmodified RNA is able to catalyse Diels-Alder reactions with aromatic dienes [4]. In the selection, each molecule of the RNA library was coupled via a poly(ethy1ene glycol) tether to anthracene as the diene, while the other reactant (maleimide as the dienophile) was biotinylated (Figure 1A). After ten rounds of selection and amplification, 16 independent sequence families were found to significantly accelerate the Diels-Alder reaction. Thirteen of these families contained a small motif, which was found to be responsible for catalysis. A 49-mer RNA containing this motif accelerates the reaction between the tethered anthracene and biotin male-

Figure I RNA-catalysed Diels-Alder reaction (A) Reaction ofa tethered aromatic diene with biotin rnaleimide. (6)Secondary structure motif responsible for catalysis.

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Ribozymes and RNA Catalysis

Figure 2

tween different enantiomers of chiral substrates and accelerates cycloadditions with both enantioand diastereoselectivity. T h e stereochemistry of the reaction is controlled by RNA-diene interactions. T h e RNA interacts strongly and stereoselectively with the cycloaddition products, requiring several structural features to be present. Strong and stereoselective product inhibition is observed. Taken together, the results highlight the intricacy of ribozyme active sites which can control chemical reaction pathways, based on minute differences in substrate stereochemistry and substitution pattern [6].

Ribozyme-mediated catalysis in trans of a Diels-Alder reaction 0

+ +R. 0

Mutation analysis of the Diels-Alderase ribozymes In a systematic study, we investigated the roles of individual nucleotides in the formally singlestranded regions, and probed the existence of the double-stranded structural elements. These studies confirmed the structural proposals made in Figure l(B), and two additional tertiary interactions were found. Currently, we are extending these studies to individual atoms and functional groups (D. Bebenroth, B. Seelig and A. Jaschke, unpublished work).

enantioselectivity was shown to be dependent primarily on the size of the substituent at the anthracene ring system. Finally, in the course of the stereochemical investigations, a ‘mirror image’ of the ribozyme was synthesized from L-nucleotides and was demonstrated to be catalytically active. This L-ribozyme showed, as expected, exactly the opposite enantioselectivity [S] . Thus these DielsAlderase ribozymes show several features of enzymic catalysis which were not previously demonstrated for RNA.

Mechanistic considerations At present, it is unclear how RNA achieves catalysis of C-C bond formation. It is generally agreed that, in a Diels-Alder reaction, bond formation occurs by concerted mixing of the highest-occupied molecular orbital of the diene and the lowest-unoccupied molecular orbital (LUMO) of the dienophile. T h e majority of small molecule (Lewis acid) catalysts operate by coordination with (and withdrawal of electron density from) the dienophile, thereby lowering the energy of its L U M O [7]. For antibody catalysis of a Diels-Alder reaction, both binding of the reactants in a reactive conformation and lowering the dienophile’s L U M O due to hydrogen bonding to the dienophile carbonyl oxygen have been discussed on the basis of X-ray crystallographic investigations [8,9]. A theoretical study found that catalytic efficiency is achieved by enthalpic stabilization of the transition state, near-perfect shape complementarity of binding site and transition state, and a strategically placed hydrogen bond [lo]. It is conceivable that similar mechanisms are involved in ribozyme-mediated catalysis as well. T h e Houk group recently compared several artificial catalytic Diels-Alderase systems, in-

Interactions between a Diels-Alderase ribozyme and i t s substrates and products T h e interactions of these Diels-Alderase ribozymes with their substrates and products have been elucidated by chemical substitution analysis using 44 different, systematically varied analogues [6]. RNA-diene interaction is governed by stacking interactions, whereas hydrogen bonding and metal ion co-ordination appear to be less important. T h e diene has to be an anthracene derivative, and substituents at defined positions are permitted, thereby shedding light on the geometry of the binding site. Interestingly, the poly(ethy1ene glycol) tether used in the selection does not make any contribution to binding and can be removed without penalty. T h e dienophile must be a five-membered maleimidyl ring with an unsubstituted reactive double bond, and a hydrophobic side chain makes a major contribution to RNA binding. T h e ribozyme distinguishes be-

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Biochemical Society Transactions (2002) Volume 30, part 6

cluding our ribozymes, and computed catalytic efficiencies and AG values [ l l ] . While this ribozyme compared favourably with several catalytic antibodies and other artificial systems, their study concluded that, in contrast with naturally evolved and fine-tuned enzyme, there is no significant specific stabilization of the transition state in any of these artificial systems. According to that study [ 113, acceleration predominantly arises from binding of reactants, converting a second-order reaction of diene with dienophile into a firstorder reaction of the termolecular complex of host, diene, and dienophile, rather than from a special binding of transition states. T h e simultaneous presence of the two components of an intermolecular Diels-Alder reaction within the confined space of a cavity is the driving force that facilitates the reaction [ 111.

molecular basis of these interactions is far from being understood.

References I Jaschke,A. (200 I ) Biol. Chern. 382, I 32 I - I 325 2 Morns, K. N.,Tarasow, T. M., julin. C. M., Sirnons, S. L., Hilvert, D. and Gold, L. (1994) Proc. Natl. Acad. Sci. U.S.A. 9 I, I 3028- I 3032 3 Tarasow, T. M., Tarasow. S. L. and Eaton, 6. E. ( I 997) Nature (London) 389,54-57 4 Seelig, 6. and Jaschke,A. ( 1999) Chem. Biol. 6, 167- I76 5 Seelig, B., Keiper, S., Stuhlmann, F. and Jaschke,A. (2000) Angew. Chem. Int. Ed. Engl. 39,4576-4579 6 Stuhlmann, F. and Jaschke,A. (2002) J. Am. Chern. SOC. 124, 3238-3244 7 Tarasow, T. M. and Eaton, 6. E. ( I 999) Cell. Mot. Life Sci. 55, 1463- I472 8 Heine, A., Stura, E. A., Yli-Kauhaluoma,J , T., Gao, C., Deng, Q.. Beno, 6. R., Houk. K. N.. Janda,K. D. and Wilson, I. A. ( 1998) Science 279, 1934- I940 9 Romesberg, F. E., Spiller, B., Schultz P. G. and Stevens, R. C. ( I 998) Science 279, 1929- I933 10 Chen, J., Deng, Q,,Wang, R., Houk, K. N. and Hilvert, D. (2000) Chembiochem I, 255-26 I I I Kim, S. P., Leach, A. G. and Houk, K. N. (2002) J. Org. Chem. 67,4250-4260

Conclusions Despite their limited set of functional groups, ribozymes can accelerate complex organic transformations such as Diels-Alder reactions between small molecules in a way similar to that of protein enzymes or traditional chemical catalysts, with multiple turnover and stereoselectivity. T h e

Received 20 August 2002

lntracellular ribozyme applications

D.CastanOtto*, J. R. Li*. A. Michienzi*, M.-A. Langloist. N. S. Lee*, J. Puyrniratt and J. J. Rossi*' *Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, C A 9 I0 10-30I I , U.S.A., and t Laboratory of Human Genetics, Lava1 University Medical Research Centre, CHUQ, Pavillon CHUL, Ste-Foy, Quebec, Canada G I V 7P4 Abstract

sible to ribozyme base pairing. Cellular proteins greatly influence the trafficking and structure of RNA, and therefore making ribozymes work effectively in cells a significant challenge. This article addresses the problems of getting engineered ribozymes to effectively pair with and cleave targets in cells. T h e work described here illuminates methods for target-site selection on native mRNAs, methods for ribozyme expression, and strategies for obtaining a discrete intracellular localization of ribozymes.

T h e exquisite target selectivity of trans-acting ribozymes has fostered their use as potential therapeutic agents and tools for down-regulating cellular transcripts. In living cells, free diffusion of RNAs is extremely limited, if it exists at all. Thus, getting ribozymes to base-pair with their cognate targets requires co-localizing the ribozyme transcript with the target RIVA. In addition, not all sites along a given target RNA are equally acces-

Introduction Key words cancer, functional genomics. genetic disease, HIV, nucleolus Abbreviations used DMPK. dystrophica rnyotonica-protein kinase MDM2, murtne double minute 2 'To whom correspondence should be addressed (e-mail jrossi(a bncoh.edu)


Ribozymes are RNA molecules that are capable of acting as enzymes, even in the complete absence of proteins. They have the catalytic activity of breaking and/or forming covalent bonds with extraordinary specificity, thereby accelerating the rate

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