The Catalytic Spectrum of Ribozymes

Seven natural types of oligonucleotides with catalytic properties are known: hammerhead, hairpin, hepatitis delta virus, ribonuclease P, varkud satellite ribozyme, group I intron, and group II intron (Doudna and Cech, 2002). Catalysis through natural ribozymes is restricted to hydrolysis and transesterification reactions at in-

ternucleotide phosphodiester bonds, whereas artificial ribozymes can also accelerate miscellaneous types of chemical reactions. These reactions include acylation (Illangasekare et al., 1995; Lohse and Szostak, 1996; Jenne and Famulok, 1998), alkylation (Wilson and Szostak, 1995), formation of amide bonds (Wiegand et al., 1997; Zhang and Cech, 1997), C-C bonds (Diels-Alder reaction) (Tarasow et al., 1997; Seelig and Jäschke, 1999; Seelig et al., 2000; Stuhlmann and Jäschke, 2002), C-S bonds by Michael addition (Sengle et al., 2001), N-glycosidic bonds (Unrau and Bartel, 1998), and even a redox reaction (Tsukiji et al., 2003).

In the following, the scope of in vitro selected ribozymes will be pointed out with special regard to organic chemistry. Several highlights will be discussed in detail. In Table 9.1 the variety of reactions catalyzed by ribozymes, including some interesting DNAzymes, is summarized.

Table 9.1 Catalytic activities of ribozymes

Reaction type Equation

References

C-C bonds

Diels-Alder *

reaction +

Biphenyl isomerization

Biphenyl isomerization

Seelig and Jäschke, 1999; Seelig et al., 2000; Stuhlmann and Jäschke, 2002; Tarasow et al., 2004; Tarasow et al., 1997

Prudent et al., 1994

C-N bonds

Amide bond formation

N R2 H2

N-Glycosidic bond formation

N-Alkylation

N R2 H2

Baskerville and Bartel, )H 2002; Sun et al., 2002;

Wiegand et al., 1997; Zhang and Cech, 1997 R1 r2 Chapple et al., 2003;

Unrau and Bartel, 1998

Wilson and Szostak, 1995

depurination

depurination qh hn

Sheppard et al., 2000a

r qh hn

Table 9.1 Catalytic activities of ribozymes (continued)

Reaction type Equation

References

C-S bonds

S-Michael reaction

S-Acylation S-Alkylation o r n h nhr1

R1 OH

OP S OR

Sengle et al., 2001

Jadhav and Yarus, 2002

O Wecker et al., 1996

C-O bonds

Transesterification

Carbonate ester hydrolysis

RO R2 R1 = small oligomer, AMP

R1O OR.

Illangasekare et al., 1995; Illangesekare and Yarus, 1999 Lohse and Szostak, 1996 Jenne and Famulok, 1998 Lee et al., 2000; Murakami et al., 2003 Chun et al., 1999

o o r nhr1

R SH

R1O R

RS R

Ri OH

R1O R

P-O bonds

Phospho-anhydride formation

Phosphorylation r—oh + NTP

Lorsch and Szostak, 1994

Johnston et al., 2001 Ekland et al., 1995; Kuhne and Joyce, 2003; Landweber and Pokrovskaya, 1999; McGinness and Joyce, 2002; Roger and Joyce, 1999

Carmi et al., 1996a Landweber and Pokrovskaya, 1999; Lazarev et al., 2003 Feldman and Sen, 2001; Santoro and Joyce, 1997; Santoro and Joyce, 1998; Santoro et al., 2000a Kong et al., 2002b r p r

Table 9.1 Catalytic activities of ribozymes (continued)

Reaction type

Equation

References

C-metal bonds

Porphyrin metallation

Ri Ri

Conn et al., 1996 Kawazoe et al., 2001; Li and Sen, 1996a

Redox reactions

Oxidation and reduction

R OH ^

R^H

Tsukiji et al., 2003; Tsukiji et al., 2004

R generally indicates nucleic acid containing residue; R1_2, various moieties; Hal, halogen; XP, phosphate; NDP, nucleoside diphosphate; NTP, nucleoside triphosphate; a DNA enzymes, b RNA-DNA enzymes.

R generally indicates nucleic acid containing residue; R1_2, various moieties; Hal, halogen; XP, phosphate; NDP, nucleoside diphosphate; NTP, nucleoside triphosphate; a DNA enzymes, b RNA-DNA enzymes.

The catalytic spectrum of RNA cannot be discussed without the background of a hypothetical prebiotic RNA world. As stated in the introduction, the concept of an RNA world means that RNA, at one point, may have performed almost all catalytic functions necessary for survival. As Woese points out (Woese, 2002), the term "survival" does not necessarily apply to a discrete, living entity or even to distinct species, be they single molecules or single cells. Therefore, and because it is unclear exactly which chemical reactions might have been catalyzed by RNA or other matter such as catalytic surfaces (Miyakawa and Ferris, 2003), a complete picture of the RNA's catalytic potential does not come with the RNA world hypothesis. Yet, there are a large number of chemical reactions and properties that have been predicted to occur in an RNA world. For many of the most important reactions, proof of the catalytic potential has been given, including for example ribo-nucleotide polymerization, aminoacylation, and peptide bond formation.

Although the RNA world is thought to have existed almost four billion years ago, X-ray-structures of the ribosome (e.g. Nissen et al., 2000; Moore and Steitz, 2003) seem to exhibit fragments of this ancestral era. The structures revealed that RNA-mediated catalysis plays an important role in the peptide synthesis of the ri-bosome. The key step in translation is catalyzed only by the ribonucleic acid component of the ribosome, without any direct contribution of proteins from the spatial vicinity. This impressively demonstrates the catalytic potential of RNA in a biochemical reaction that may arguably be called the most important ever.

An artificial ribozyme has been shown to mimic this translation step of the ri-bosome (Zhang and Cech, 1997). The specificity of this selected ribozyme is based on the recognition of an adenosine moiety of the amino acid ester and allows the utilization of leucine- and phenylalanine- as well as methionine-derivatized substrates. This tolerance for various amino acids indicates the possibility of selecting more general ribozymes for protein synthesis. Furthermore, a recently characterized ribozyme efficiently catalyzes the synthesis of 30 different dipeptides from an aminoacyl-adenylate substrate. Ribozyme-mediated synthesis of uncoded peptides may have been an important step in the transition from a RNA to a peptide world before the emergence of the ribosome (Sun et al., 2002).

Further hints supporting the existence of an RNA world could be the isolation of ribozymes which perform and accelerate intermolecular ligation of the 3'-hy-droxyl group of various oligonucleotides to the 5'-triphosphate of an RNA hairpin (McGinness and Joyce, 2002) and the in trans-aminoacylation of a tRNA with specific recognition of an activated amino acid (Lee et al., 2000; Murakami et al., 2003).

Another important development was the isolation of a ribozyme which performs nucleotide synthesis by forming a glycosidic linkage from activated ribose (pRpp) (Unrau and Bartel, 1998) in a way similar to the modern biosynthesis of nucleotides.

Before the discovery of catalytic RNA, the principal indications of a possible RNA world had been the role of tRNA and ribosomal RNA in translation, the use of RNA as genetic material in retroviruses, and the ubiquitous occurrence of RNA-related enzymatic cosubstrates such as GTP, ATP, AMP, cAMP, SAM, FADH2, and NAD+ in all major metabolic pathways. Clearly, the ability of RNA to employ these and other ubiquitous cosubstrates in catalysis must be expected.

A recent highlight in ribozyme research is the in vitro evolution of a ribozyme that oxidizes an alcohol in a NAD+-dependent manner. The resulting RNA-alde-hyde was trapped via a chemoselective modification with biotin hydrazide. The function of this ribozyme is analogous to the natural alcohol dehydrogenase enzyme (ADH) and depends on the same cofactors (Tsukiji et al., 2003). Furthermore this ribozyme was coupled with an electron transfer process between NADH and FAD. Thus a NAD+ regeneration system is constituted. Interestingly the reverse reaction, the RNA-catalyzed reduction of the aldehyde, is also possible in the presence of NADH (Fig. 9.2) (Tsukiji et al., 2004). This is the first clear-cut demonstration of a typical redox reaction catalyzed by RNA.

Further artificial ribozymes are known to react with cosubstrates, for example acylating the thiol group of tethered coenzyme A with the AMP-activated biotin. These ribozymes also produce the crucial metabolic intermediates acetyl-CoA and butyryl-CoA at substantial reaction rates. For the selection of this ribozyme, the employed RNA pool had been coupled at its 5'-end to CoA by a previously isolated capping ribozyme (Jadhav and Yarus, 2002).

The current hypothesis predicts evolution of the RNA world into the modern DNA-RNA-protein world, with DNA taking over the role of storage of genomic information. One advantage of DNA over RNA as genetic material is the better chemical and enzymatic stability of deoxynucleic acids. That same advantage is also shown by deoxyribozymes, a wide range of which have now been selected. Despite this, there is little evidence that ribozymes from the RNA world have

Fig. 9.2 Reactions performed by an in vitro selected redox-active ribozyme. The redox reaction is NAD+/NADH-dependent, similar to natural alcohol dehydrogenase enzymes. The

Ribozyme oxidation of the benzyl alcohol can also be coupled to a spontaneous electron transfer between NADH and FAD (Tsukiji et al., 2003, 2004).

Fig. 9.2 Reactions performed by an in vitro selected redox-active ribozyme. The redox reaction is NAD+/NADH-dependent, similar to natural alcohol dehydrogenase enzymes. The

Ribozyme oxidation of the benzyl alcohol can also be coupled to a spontaneous electron transfer between NADH and FAD (Tsukiji et al., 2003, 2004).

been replaced by deoxyribozymes. Rather, proteins have taken over the vast majority of catalytic functions. Because deoxyribozymes have little relevance to organic chemistry, these will not be discussed here in detail.

The intrinsically restricted functionality of nucleic acids compared with that of proteins is a serious shortcoming for the expression of catalytic potential. A possible remedy is the introduction of additional, non-natural functional groups via the incorporation of modified nucleotides (reviewed by Verma et al., 2003). The tolerance of the employed RNA polymerase towards the modifications limits the general use of this technique for the generation of modified ribozymes. Many such ribozymes with modified bases catalyze RNA cleavage or ligation (Beaudry et al., 2000; Dai and Joyce, 2000; Santoro et al., 2000; Teramoto and Joyce, 2000). Further catalytic activities concern metallation of N-methylmesopor-phyrin (Kawazoe et al., 2001), formation of a phosphodiester bond with a deoxy-nucleotide (Teramoto and Joyce, 2000), and cleavage of phosphodiester in trans under simulated physiological conditions (Zinnen et al., 2002).

The incorporation by T7 RNA polymerase and successful use in SELEX of a number of uridine derivatives has been reported by Eaton's laboratory (Vaught et al., 2004), resulting in the development of a cupric ion-dependent modified ribozyme with Diels-Alderase activity (Tarasow et al., 1997, 1999, 2004). As far as we know, the Diels-Alder reaction does not play a major role in any biochemical pathway, and the use of modified nucleotides to expand the catalytic repertoire of RNA was certainly not undertaken with the RNA world as principal motivation. Rather, these approaches underscore the potential for application of catalytic RNA in modern fields of biotechnology and organic synthesis. The use of proteic enzymes in organic synthesis has become a commonplace strategy where applic able, the limiting factor being the choice of suitable enzymes and their respective substrate specificities. From this perspective, several ribozymes display catalytic activity of high interest to the organic chemist (see Table 9.1). Importantly, most of these ribozymes have been obtained using a direct selection protocol, where the choice of reactants has dictated the substrate specificity of the resulting ribozymes.

Thus, the direct selection method holds the alluring possibility of engineering substrate-specific ribozymes that are tailored to the particular reaction an organic chemist might wish to carry out. Since nucleic acids are chiral, one can even anticipate stereoselectivity in the would-be custom-made catalysts. Although this remains a vision yet to be accomplished, several cases of ribozymes with promising properties have been reported, accelerating certain redox reactions, Michael additions, and cycloadditions. The oxidation of a benzyl alcohol to the corresponding aldehyde has already been mentioned, and the reverse reaction from Suga's laboratory. The redox ribozymes still require their substrate to be covalently bound, meaning they act in cis, thus performing neither true catalysis nor multiple turnover (Tsukiji et al., 2003).

In the Famulok laboratory, a ribozyme has been isolated that promotes a reaction corresponding to the first step of the formation of dTMP from dUMP in pro-teic thymidylate synthases. This ribozyme mediates Michael-adduct formation at a Michael-acceptor substrate. The reaction is accelerated by a factor of nearly 105. The selected ribozyme could be engineered to act in an intermolecular reaction on a substrate tethered to an RNA oligomer. The demonstration of RNA catalysis of this reaction has bearing on the RNA world hypothesis, as well as implications for possible synthetic applications (Eisenführ et al., 2003; Sengle et al., 2001).

Among the several routes for the creation of ribozymes for a Diels-Alder reaction, a minimal conserved motif (Fig. 9.3b) from a direct selection with linkercoupled reactants conducted in our laboratory so far displays the most promising properties for a potential application in organic synthesis. This construct, a 49-mer RNA, accelerates the formation of C-C bonds between RNA-tethered anthracene and biotinylated N-alkylmaleimides by a factor of up to 18 500. The 49-mer motif features 11 conserved nucleotides in a bulge region and variable helical stems. In contrast to other ribozymes that accelerate bond-forming reactions (Illangasekare et al., 1995; Jenne and Famulok, 1998; Tarasow et al., 1997; Wiegand et al., 1997), this Diels-Alderase behaves like a typical protein enzyme displaying Michaelis-Menten kinetics and works in a bimolecular manner on two reactants that are not attached to any RNA. True catalysis with multiple turnover is performed with a kcat of about 20 min-1. This Diels-Alderase was employed in the first-ever demonstration of RNA-catalyzed enantioselective transformations. The minimal motif yields the Diels-Alder product as a single enantiomer with an enantioselectivity of up to 95% ee, depending on the substitution pattern of the anthracene. Use of the mirror-image l-ribozyme leads to the opposite enantiomer, whereas the uncatalyzed reaction yields a racemic mixture (Seelig et al., 2000) (Fig. 9.3b). Systematic variation of diene and dienophile showed that the Diels-Alderase distinguishes between different enantiomers of achiral substrates and

Fig. 9.3 Ribozyme-catalyzed Diels-Alder reaction in trans. (a) Reaction equation. (b) Reciprocal enantioselectivity in the Diels-Alder reactions catalyzed by the natural d-ribozyme and the spiegelmer (l-ribozyme). Proposed secondary structure motif of the Diels-Alderase ribozymes. The chromatograms show the analysis by chiral high-performance liquid

Fig. 9.3 Ribozyme-catalyzed Diels-Alder reaction in trans. (a) Reaction equation. (b) Reciprocal enantioselectivity in the Diels-Alder reactions catalyzed by the natural d-ribozyme and the spiegelmer (l-ribozyme). Proposed secondary structure motif of the Diels-Alderase ribozymes. The chromatograms show the analysis by chiral high-performance liquid chromatography (HPLC) of reaction products generated by d-RNA (left) and by l-RNA (right) (R, = (C2H4O)6-H, R2 = (CH2)5C00CH3). The two peaks correspond to the two enantiomers. The ribozyme-catalyzed reaction produces one enantiomer in a 20-fold excess whereas the uncatalyzed background (light gray line) shows a racemic (1:1) mixture (Seelig et al., 2000).

hence displays diastereoselectivity (Stuhlmann and Jäschke, 2002). The dieno-phile has to be a five-membered maleimidyl ring without substitution at the double bond. A hydrophobic side chain contributes to ribozyme binding.

Fig. 9.4 Architecture of the Diels-Alder ribozyme (Keiper et al., 2004). (a) Refined secondary structure. (b) Modeled three-dimensional structure.

Some insight into the tertiary structure has been obtained from a variety of probing experiments and a comprehensive mutation analysis. The postulated working model features a Y-shaped framework of double helical arms including an asymmetric internal loop. In an unprecedented manner, nucleotides of the 5'-terminus clamp the opposite sides of the bulge, forming a double pseudo-knot. These investigations also suggest that in this rigid structure no significant conformational changes occur upon binding of substrates or products during the catalytic process (Keiper et al., 2004) (Fig. 9.4).

A feature of interest to both biotechnology and organic synthesis is the persistence of catalytic activity upon immobilization, which has been unambiguously demonstrated using both enantiomers of the ribozyme. Products of the respective stereochemistry have been obtained after incubation of the reactants with solid phase onto which either enantiomeric ribozyme had been conjugated. The resins remained active even after storage and use for several months, opening up the possibility of the development of a ribozyme reactor prototype for economic synthesis of Diels-Alder products (Schlatterer et al., 2003). Similar stability of an immobilized RNA was reported for an aminoacyl-tRNA synthesis ribozyme, which retained its activity after five cycles (Murakami et al., 2002).

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