Download Nucleic acid enzymes

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 ([email protected])
Current Opinion in Biotechnology 2005, 16:614–621
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
Current Opinion in Biotechnology 2005, 16:614–621
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 Zn2+
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
Zn2+, 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 30 -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 50 and the 30 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 4SU 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 reblock-
ing/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 4SU
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 50 - and 30 primer sequences at the corresponding end (108 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
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
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 45nucleotide-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 30
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 20 -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
Current Opinion in Biotechnology 2005, 16:614–621
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 Cu2+ and now, in a
mutated sequence, of Cu2+ and Ni2+ [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 Km) 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 30 –50 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 30 -OH of one
RNA strand and the 50 -triphosphate of the other, the
nascent ligation junction was embedded within a duplex
(DNA:RNA) region. These catalysts are relatively slow
www.sciencedirect.com
(kobs = 0.008 min1 for the most active clone) but specifically 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 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 30 –50 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 30 –50 or 20 –50 ; Figure 4)
strongly depend on the substrate employed during the
selection. This would be a very unfortunate occurrence in
the search for generally applicable 30 –50 ligase DNAzymes. It was actually possible, however, to shift the
selection outcome towards the desired 30 –50 linkage by
implementing a cleavage step with an 8–17 DNAzyme
[36,37]. In this way, all sequences catalyzing the correct
30 –50 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 Zn2+
for catalytic activity has also been reported [38]. In the
presence of 1 mM Zn2+ these catalysts promote the formation of a variety of RNA linkages: linear 30 –50 (not
obtained in the analogous selection in the presence of
Mg2+), 20 –50 , 30 –20 , 20 –20 , and branched 20 –20 (30 ).
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 kobs as high as 0.01 min1 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.
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
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
Current Opinion in Biotechnology 2005, 16:614–621
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 RiboReporterTM 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 metalwww.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: Mg2+,
K+, Li+, Na+, Rb+, Ca2+, Sr2+, Cd2+, Co2+, Mn2+, Ni2+ and
Zn2+, whereas only Mg2+ was present during the counterselection. Five different classes of ribozymes were isolated from this selection with significantly increased
activity in the presence of Cd2+, Co2+, Mn2+, Ni2+ and
Zn2+. None of the selected ribozymes was found to
respond to Ca2+, Sr2+ 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,
www.sciencedirect.com
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.
Current Opinion in Biotechnology 2005, 16:614–621
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 107. 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/Zn2+-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 5phosphoribosyl-1-pyrophosphate to give 6-thioguanine monophosphate
with apparent efficiency (kcat app/Km) as high as 284 M1 min1.
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 30 -50 RNA
linkages. J Am Chem Soc 2004, 126:16426-16432.
35. Wang Y, Silverman SK: Directing the outcome of
deoxyribozyme selections to favor native 30 -50 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 30 –50 linkages, gave, after only four additional
selection cycles, 23 out of 24 isolated clones with the desired 30 – 50 ligase
activity.
19. Unrau PJ, Bartel DP: RNA-catalyzed nucleotide synthesis.
Nature 1998, 395:260-263.
36. Cruz RPG, Withers JB, Li Y: Dinucleotide junction cleavage
versatility of 8-17 deoxyribozyme. Chem Biol 2004,
11:57-67.
20. Chapple KE, Bartel DP, Unrau PJ: Combinatorial minimization
and secondary structure determination of a nucleotide
synthase ribozyme. RNA 2003, 9:1208-1220.
37. Santoro SW, Joyce GF: A general purpose RNA-cleaving DNA
enzyme. Proc Natl Acad Sci USA 1997, 94:4262-4266.
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.
38. Hoadley KA, Purtha WE, Wolf AC, Flynn-Charlebois A,
Silverman SK: Zn2+-Dependent deoxyribozymes that form
natural and unnatural RNA linkages. Biochemistry 2005,
44:9217-9231.
39. Liu J, Lu Y: A colorimetric lead biosensor using DNAzyme
directed 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 Pb2+.
40. Thomas JM, Ting R, Perrin DM: High affinity DNAzyme-based
ligands for transition metal cations — a prototype sensor for
Hg2+. 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.
27. Baram D, Yonath A: From peptide-bond formation to
cotranslational folding: dynamic, regulatory and evolutionary
aspects. FEBS Lett 2005, 579:948-954.
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.
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.
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
Current Opinion in Biotechnology 2005, 16:614–621
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
M2+-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.
www.sciencedirect.com
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 bgalactosidase 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.
Current Opinion in Biotechnology 2005, 16:614–621