Deoxyribozymes, also called
DNA enzymes, DNAzymes, or catalytic DNA,
DNA oligonucleotides that are capable of performing a specific
chemical reaction, often but not always catalytic. This is similar to
the action of other biological enzymes, such as proteins or ribozymes
(enzymes composed of RNA). However, in contrast to the abundance of
protein enzymes in biological systems and the discovery of biological
ribozymes in the 1980s, there are no known naturally occurring
deoxyribozymes. Deoxyribozymes should not be confused with DNA
aptamers which are oligonucleotides that selectively bind a target
ligand, but do not catalyze a subsequent chemical reaction.
With the exception of ribozymes, nucleic acid molecules within cells
primarily serve as storage of genetic information due to its ability
to form complementary base pairs, which allows for high-fidelity
copying and transfer of genetic information. In contrast, nucleic acid
molecules are more limited in their catalytic ability, in comparison
to protein enzymes, to just three types of interactions: hydrogen
bonding, pi stacking, and metal-ion coordination. This is due to the
limited number of functional groups of the nucleic acid monomers:
while proteins are built from up to twenty different amino acids with
various functional groups, nucleic acids are built from just four
chemically similar nucleobases. In addition,
DNA lacks the 2'-hydroxyl
group found in
RNA which limits the catalytic competency of
deoxyribozymes even in comparison to ribozymes.
In addition to the inherent inferiority of
DNA catalytic activity, the
apparent lack of naturally occurring deoxyribozymes may also be due to
the primarily double-stranded conformation of
DNA in biological
systems which would limit its physical flexibility and ability to form
tertiary structures, and so would drastically limit the ability of
DNA to act as a catalyst; though there are a few
known instances of biological single-stranded
DNA such as multicopy
DNA (msDNA), certain viral genomes, and the
replication fork formed during
DNA replication. Further structural
RNA may also play a role in the lack of
biological deoxyribozymes, such as the additional methyl group of the
DNA base thymidine compared to the
RNA base uracil or the tendency of
DNA to adopt the B-form helix while
RNA tends to adopt the A-form
helix. However, it has also been shown that
DNA can form structures
RNA cannot, which suggests that, though there are differences in
structures that each can form, neither is inherently more or less
catalytic due to their possible structural motifs.
1.3 Other reactions
2.1 in vitro selection
2.2 in vitro evolution
3 "True" catalysis?
4.1 Drug clinical trials
4.3 Asymmetric synthesis
4.4 Other uses
5 See also
7 External links
The trans-form (two separate strands) of the 17E DNAzyme. Most
ribonuclease DNAzymes have a similar form, consisting of a separate
enzyme strand (blue/cyan) and substrate strand (black). Two arms of
complementary bases flank the catalytic core (cyan) on the enzyme
strand and the single ribonucleotide (red) on the substrate strand.
The arrow shows the ribonucleotide cleavage site.
The most abundant class of deoxyribozymes are ribonucleases, which
catalyze the cleavage of a ribonucleotide phosphodiester bond through
a transesterification reaction, forming a 2'3'-cyclic phosphate
terminus and a 5'-hydroxyl terminus.
typically undergo selection as long, single-stranded oligonucleotides
which contain a single ribonucleotide base to act as the cleavage
site. Once sequenced, this single-stranded "cis"-form of the
deoxyribozyme can be converted to the two-stranded "trans"-form by
separating the substrate domain (containing the ribonucleotide
cleavage site) and the enzyme domain (containing the catalytic core)
into separate strands which can hybridize through two flanking arms
consisting of complementary base pairs.
The first known deoxyribozyme was a ribonuclease, discovered in 1994
Ronald Breaker while a postdoctoral fellow in the laboratory of
Gerald Joyce at the Scripps Research Institute. This deoxyribozyme,
later named GR-5, catalyzes the Pb2+-dependent cleavage of a single
ribonucleotide phosphoester at a rate that is more than 100-fold
compared to the uncatalyzed reaction. Subsequently, additional
RNA-cleaving deoxyribozymes that incorporate different metal cofactors
were developed, including the Mg2+-dependent E2 deoxyribozyme and
the Ca2+-dependent Mg5 deoxyribozyme. These first deoxyribozymes
were unable to catalyze a full
RNA substrate strand, but by
incorporating the full
RNA substrate strand into the selection
process, deoxyribozymes which functioned with substrates consisting of
RNA or full
DNA with a single
RNA base were both able to
be utilized. The first of these more versatile deoxyribozymes,
8-17 and 10-23, are currently the most widely studied deoxyribozymes.
In fact, many subsequently discovered deoxyribozymes were found to
contain the same catalytic core motif as 8-17, including the
previously discovered Mg5, suggesting that this motif represents the
"simplest solution for the
RNA cleavage problem". The 10-23
DNAzyme contains a 15-nucleotide catalytic core that is flanked by two
substrate recognition domains. This DNAzyme cleaves complementary RNAs
efficiently in a sequence specific manner between an unpaired purine
and a paired pyrimidine. DNAzymes targeting AU or GU vs. GC or AC are
more effective. Furthermore, the
RNA cleavage rates have been shown to
increase after the introduction of intercalators or the substitution
of deoxyguanine with deoxyinosine at the junction of the catalytic
loop. Specifically, the addition of 2’-O-methyl modifications to the
catalytic proved to significantly increase the cleavage rate both in
vitro and in vivo.  Other notable deoxyribozyme ribonucleases are
those that are highly selective for a certain cofactor. Among this
group are the metal selective deoxyribozymes such as Pb2+-specific
17E, UO22+-specific 39E, and Na+-specific A43. First
crystal structure of a DNAzyme was reported in 2016.
Of particular interest are
DNA ligases. These molecules have
demonstrated remarkable chemoselectivity in
RNA branching reactions.
Although each repeating unit in a
RNA strand owns a free hydroxyl
DNA ligase takes just one of them as a branching starting
point. An accomplishment unattainable with traditional organic
Many other deoxyribozymes have since been developed that catalyze DNA
DNA deglycosylation, porphyrin
metalation, thymine dimer photoreversion and
Main article: Systematic evolution of ligands by exponential
in vitro selection
Because there are no known naturally occurring deoxyribozymes, most
known deoxyribozyme sequences have been discovered through a
high-throughput in vitro selection technique, similar to
SELEX. in vitro selection utilizes a "pool" of a large number
DNA sequences (typically 1014–1015 unique strands) that
can be screened for a specific catalytic activity. The pool is
synthesized through solid phase synthesis such that each strand has
two constant regions (primer binding sites for PCR amplification)
flanking a random region of a certain length, typically 25–50 bases
long. Thus the total number of unique strands, called the sequence
space, is 4N where N denotes the number of bases in the random region.
Because 425 ≈ 1015, there is no practical reason to choose random
regions of less than 25 bases in length, while going above this number
of bases means that the total sequence space cannot be surveyed.
However, since there are likely many potential candidates for a given
catalytic reaction within the sequence space, random regions of 50 and
even higher have successfully yielded catalytic deoxyribozymes.
The pool is first subjected to a selection step, during which the
catalytic strands are separated from the non-catalytic strands. The
exact separation method will depend on the reaction being catalyzed.
As an example, the separation step for ribonucleotide cleavage often
utilizes affinity chromatography, in which a biological tag attached
DNA strand is removed from any catalytically active strands
via cleavage of a ribonucleotide base. This allows the catalytic
strands to be separated by a column that specifically binds the tag,
since the non-active strands will remain bound to the column while the
active strands (which no longer possess the tag) flow through. A
common set-up for this is a biotin tag with a streptavidin affinity
column. Gel electrophoresis based separation can also be used
in which the change in molecular weight of strands upon the cleavage
reaction is enough to cause a shift in the location of the reactive
strands on the gel. After the selection step, the reactive pool is
amplified via polymerase chain reaction (PCR) to regenerate and
amplify the reactive strands, and the process is repeated until a pool
of sufficient reactivity is obtained. Multiple rounds of selection are
required because some non-catalytic strands will inevitably make it
through any single selection step. Usually 4–10 rounds are required
for unambiguous catalytic activity, though more rounds are often
necessary for more stringent catalytic conditions. After a sufficient
number of rounds, the final pool is sequenced and the individual
strands are tested for their catalytic activity. The dynamics of
the pool can be described through mathematical modeling  , which
shows how oligonucleotides undergo competitive binding with the
targets and how the evolutionary outcome can be improved through fine
tuning of parameters.
Deoxyribozymes obtained through in vitro selection will be optimized
for the conditions during the selection, such as salt concentration,
pH, and the presence of cofactors. Because of this, catalytic activity
only in the presence of specific cofactors or other conditions can be
achieved using positive selection steps, as well as negative selection
steps against other undesired conditions.
in vitro evolution
A similar method of obtaining new deoxyribozymes is through in vitro
evolution. Though this term is often used interchangeably with in
vitro selection, in vitro evolution more appropriately refers to a
slightly different procedure in which the initial oligonucleotide pool
is genetically altered over subsequent rounds through genetic
recombination or through point mutations. For point mutations,
the pool can be amplified using error-prone PCR to produce many
different strands of various random, single mutations. As with in
vitro selection, the evolved strands with increased activity will tend
to dominate the pool after multiple selection steps, and once a
sufficient catalytic activity is reached, the pool can be sequenced to
identify the most active strands.
The initial pool for in vitro evolution can be derived from a narrowed
subset of sequence space, such as a certain round of an in vitro
selection experiment, which is sometimes also called in vitro
reselection. The initial pool can also be derived from
amplification of a single oligonucleotide strand. As an example of the
latter, a recent study showed that a functional deoxyribozyme can be
selected through in vitro evolution of a non-catalytic oligonucleotide
precursor strand. An arbitrarily chosen
DNA fragment derived from the
RNA transcript of bovine serum albumin was evolved through random
point mutations over 25 rounds of selection. Through deep sequencing
analysis of various pool generations, the evolution of the most
catalytic deoxyribozyme strand could be tracked through each
subsequent single mutation. This first successful evolution of
DNA from a non-catalytic precursor could provide support for
RNA World hypothesis. In another recent study, an
ribozyme was converted into a deoxyribozyme through in vitro evolution
of the inactive deoxyribo-analog of the ribozyme. The new
deoxyribozyme contained just twelve point mutations, two of which had
no effect on activity, and had a catalytic efficiency of approximately
1/10 of the original ribozyme, though the researches hypothesized that
the activity could be further increased through further selection.
This first evidence for transfer of function between different nucleic
acids could provide support for various pre-
RNA World hypotheses.
Because most deoxyribozymes suffer from product inhibition and thus
exhibit single-turnover behavior, it is sometimes argued that
deoxyribozymes do not exhibit "true" catalytic behavior since they
cannot undergo multiple-turnover catalysis like most biological
enzymes. However, the general definition of a catalyst requires only
that the substance speeds up the rate of a chemical reaction without
being consumed by the reaction (i.e. it is not permanently chemically
altered and can be recycled). Thus, by this definition,
single-turnover deoxyribozymes are indeed catalysts. Furthermore,
many endogenous enzymes (both proteins and ribozymes) also exhibit
single-turnover behavior, and so the exclusion of deoxyribozymes
from the rank of "catalyst" simply because it does not feature
multiple-turnover behavior seems unjustified.
Although the discovery of
RNA enzymes predates that of
DNA enzymes the
latter have some distinct advantages.
DNA has better
DNA can be made with longer sequence length and
can be made with higher purity in Solid-phase synthesis.
Drug clinical trials
Asthma is characterized by eosinophil-induced inflammation motivated
by a type 2 helper T cell (Th2). By targeting the transcription
factor, GATA3, of the Th2 pathway, with DNAzyme it may be possible to
negate the inflammation. The safety and efficacy of SB010, a novel
10-23 DNAzyme was evaluated, and found to have the ability to cleave
and inactivate GATA3 messenger
RNA in phase IIa clinical trials.
Treatment with SB010 significantly offset both late and early
asthmatic responses after allergen aggravation in male patients with
allergic asthma. The transcription factor GATA-3 is also an
interesting target, of the DNAzyme topical formulation SB012, for a
novel therapeutic strategy in ulcerative colitis (UC). UC is an
idiopathic inflammatory bowel diseases defined by chronically
relapsing inflammations of the gastrointestinal tract, and
characterized by a superficial, continuous mucosal inflammation, which
predominantly affects the large intestine. Patients that do not
effectively respond to current UC treatment strategies exhibit serious
drawbacks one of which may lead to colorectal surgery, and can result
in a severely compromised quality of life. Thus, patients with
moderate or severe UC may significantly benefit from these new
therapeutic alternatives, of which SB012 is in phase I clinical
trials. Atopic dermatitis (AD) is a chronic inflammatory skin
disorder, in which patients suffer from eczema, often severe pruritus
on the affected skin, as well as complications and secondary
infections. AD surfaces from an upregulation of Th2-modified immune
responses, therefore a novel AD approach using DNAzymes targeting
GATA-3 is a plausible treatment option. The topical DNAzyme SB011 is
currently in phase II clinical trials. DNAzyme research for the
treatment of cancer is also underway. The development of a 10-23
DNAzyme that can block the expression of IGF-I (Insulin-like growth
factor I, a contributor to normal cell growth as well as
tumorigenesis) by targeting its m
RNA could be useful for blocking the
secretion of IGF-I from prostate storm primary cells ultimately
inhibiting prostate tumor development. Additionally, with this
treatment it is expected that hepatic metastasis would also be
inhibited, via the inhibition of IGF-I in the liver (the major source
of serum IGF-I).
DNAzymes have found practical use in metal biosensors. A
DNAzyme based biosensor for lead ion was used to detect lead ion in
water in St. Paul Public Schools in Minnesota.
Chirality is another property that a DNAzyme can exploit.
in nature as a right-handed double helix and in asymmetric synthesis a
chiral catalyst is a valuable tool in the synthesis of chiral
molecules from an achiral source. In one application an artificial DNA
catalyst was prepared by attaching a copper ion to it through a
spacer. The copper -
DNA complex catalysed a Diels-Alder reaction
in water between cyclopentadiene and an aza chalcone. The reaction
products (endo and exo) were found to be present in an enantiomeric
excess of 50%. Later it was found that an enantiomeric excess of 99%
could be induced, and that both the rate and the enantioselectivity
were related to the
Other uses of
DNA in chemistry are in DNA-templated synthesis,
DNA nanowires and
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Deoxyribozyme at the US National Library of Medicine Medical Subject
Types of nucleic acids
precursor, heterogenous nuclear
Small Cajal Body RNAs
Trans-acting small interfering