Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are
DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...
s that are capable of performing a specific
chemical reaction
A chemical reaction is a process that leads to the chemistry, chemical transformation of one set of chemical substances to another. When chemical reactions occur, the atoms are rearranged and the reaction is accompanied by an Gibbs free energy, ...
, often but not always
catalytic
Catalysis () is the increase in reaction rate, rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst ...
. This is similar to the action of other biological
enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
s, such as
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
s or
ribozyme
Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to Catalysis, catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozy ...
s (enzymes composed of
RNA
Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
).
However, in contrast to the abundance of protein enzymes in biological systems and the discovery of biological ribozymes in the 1980s,
there is only little evidence for naturally occurring deoxyribozymes.
Deoxyribozymes should not be confused with DNA
aptamers which are oligonucleotides that selectively bind a target
ligand
In coordination chemistry, a ligand is an ion or molecule with a functional group that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's el ...
, but do not catalyze a subsequent chemical reaction.
With the exception of ribozymes, nucleic acid molecules within cells primarily serve as storage of
gene
In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
tic information due to its ability to form complementary
base pair
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s, which allows for high-fidelity
copying
Copying is the duplication of information or an wiktionary:artifact, artifact based on an instance of that information or artifact, and not using the process that originally generated it. With Analog device, analog forms of information, copying is ...
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
In chemistry, a hydrogen bond (H-bond) is a specific type of molecular interaction that exhibits partial covalent character and cannot be described as a purely electrostatic force. It occurs when a hydrogen (H) atom, Covalent bond, covalently b ...
,
pi stacking, and
metal-ion coordination. This is due to the limited number of
functional group
In organic chemistry, a functional group is any substituent or moiety (chemistry), moiety in a molecule that causes the molecule's characteristic chemical reactions. The same functional group will undergo the same or similar chemical reactions r ...
s of the
nucleic acid monomers: while proteins are built from up to twenty different
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s with various functional groups, nucleic acids are built from just four chemically similar
nucleobase
Nucleotide bases (also nucleobases, nitrogenous bases) are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic building blocks of nuc ...
s. In addition, DNA lacks the 2'-
hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
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 double-stranded DNA to act as a catalyst;
though there are a few known instances of biological single-stranded DNA such as
multicopy single-stranded DNA (msDNA), certain
viral genomes, and the
replication fork formed during DNA replication. Further structural differences between DNA and RNA may also play a role in the lack of biological deoxyribozymes, such as the additional
methyl
In organic chemistry, a methyl group is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms, having chemical formula (whereas normal methane has the formula ). In formulas, the group is often abbreviated as ...
group of the DNA base
thymidine
Thymidine (nucleoside#List of nucleosides and corresponding nucleobases, symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine nucleoside, deoxynucleoside. Deoxythymidine is the DNA nuc ...
compared to the RNA base
uracil
Uracil () (nucleoside#List of nucleosides and corresponding nucleobases, symbol U or Ura) is one of the four nucleotide bases in the nucleic acid RNA. The others are adenine (A), cytosine (C), and guanine (G). In RNA, uracil binds to adenine via ...
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 that 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.
In 2021, the DNAmoreDB database for cataloguing known deoxyribozymes was released.
Types
Ribonucleases
The most abundant class of deoxyribozymes are
ribonucleases, which catalyze the
cleavage
Cleavage may refer to:
Science
* Cleavage (crystal), the way in which a crystal or mineral tends to split
* Cleavage (embryo), the division of cells in an early embryo
* Cleavage (geology), foliation of rock perpendicular to stress, a result of ...
of a
ribonucleotide phosphodiester bond
In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...
through a
transesterification
Transesterification is the process of exchanging the organic functional group R″ of an ester with the organic group R' of an alcohol. These reactions are often catalyzed by the addition of an acid or base catalyst. Strong acids catalyze the r ...
reaction, forming a 2'3'-cyclic
phosphate
Phosphates are the naturally occurring form of the element phosphorus.
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthop ...
terminus and a 5'-
hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
terminus.
Ribonuclease deoxyribozymes 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 pair
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s.
The first known deoxyribozyme was a ribonuclease, discovered in 1994 by
Ronald Breaker while a
postdoctoral
A postdoctoral fellow, postdoctoral researcher, or simply postdoc, is a person professionally conducting research after the completion of their doctoral studies (typically a PhD). Postdocs most commonly, but not always, have a temporary acade ...
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 either full 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.
Additionally, recent studies have focuses on unravelling their kinetics to further understand their performance.
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. 10-23 core based DNAzymes and the respective MNAzymes that catalyse reactions at ambient temperatures were described in 2018 and open doors for use of these nucleic acid based enzymes for many other applications without the need for heating.
A DNA molecule with sequence 5'-GGAGAACGCGAGGCAAGGCTGGGAGAAATGTGGATCACGATT-3' acts as a deoxyribozyme that uses light to repair a
thymine dimer, using
serotonin
Serotonin (), also known as 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter with a wide range of functions in both the central nervous system (CNS) and also peripheral tissues. It is involved in mood, cognition, reward, learning, ...
as
cofactor.
RNA ligases
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
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
group, the DNA ligase takes just one of them as a branching starting point. This cannot be done with traditional
organic chemistry
Organic chemistry is a subdiscipline within chemistry involving the science, scientific study of the structure, properties, and reactions of organic compounds and organic matter, organic materials, i.e., matter in its various forms that contain ...
.
Other reactions
Many other deoxyribozymes have since been developed that catalyze DNA phosphorylation, DNA
adenylation, DNA
deglycosylation,
porphyrin metalation,
thymine dimer photoreversion
and DNA cleavage.
Methods
''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 of random DNA sequences (typically 10
14–10
15 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 4
N where ''N'' denotes the number of bases in the random region. Because 4
25 ≈ 10
15, 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
Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...
, in which a
biological tag attached to each 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
Biotin (also known as vitamin B7 or vitamin H) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. ...
tag with a
streptavidin
Streptavidin is a 52 Atomic mass unit, kDa protein (tetramer) purified from the bacterium ''Streptomyces avidinii''. Streptavidin Homotetramer, homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin ...
affinity column.
Gel electrophoresis
Gel electrophoresis is an electrophoresis method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge through a gel. It is used in clinical chemistry to separate ...
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
The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...
(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
In common usage, salt is a mineral composed primarily of sodium chloride (NaCl). When used in food, especially in granulated form, it is more formally called table salt. In the form of a natural crystalline mineral, salt is also known as r ...
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
Genetic recombination (also known as genetic reshuffling) is the exchange of genetic material between different organisms which leads to production of offspring with combinations of traits that differ from those found in either parent. In eukaryot ...
or through
point mutation
A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequences ...
s.
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
mRNA transcript of
bovine serum albumin
Bovine serum albumin (BSA or "Fraction V") is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments.
The nickname "Fraction V" refers to albumin being the fifth fraction of the origin ...
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 catalytic DNA from a non-catalytic precursor could provide support for the
RNA World
The RNA world is a hypothetical stage in the evolutionary history of life on Earth in which self-replicating RNA molecules proliferated before the evolution of DNA and proteins. The term also refers to the hypothesis that posits the existence ...
hypothesis. In another recent study, an RNA ligase ribozyme was converted into a deoxyribozyme through ''in vitro'' evolution of the inactive deoxyribo-analog of the ribozyme. The new RNA ligase 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.
Applications
Although RNA enzymes were discovered before DNA enzymes, the latter have some distinct advantages. DNA is more
cost-effective
Cost-effectiveness analysis (CEA) is a form of economic analysis that compares the relative costs and outcomes (effects) of different courses of action. Cost-effectiveness analysis is distinct from cost–benefit analysis, which assigns a monetar ...
, and DNA can be made with longer sequence length and can be made with higher purity in
solid-phase synthesis
In chemistry, solid-phase synthesis is a method in which molecules are covalently bound on a solid support material and synthesised step-by-step in a single reaction vessel utilising selective protecting group chemistry. Benefits compared with ...
. Several studies have shown the usage of DNAzymes to inhibit influenza A and B virus replication in host cells.
DNAzymes have also been shown to inhibit the replication of SARS coronavirus (SARS-CoV),
Respiratory syncytial virus (RSV),
human rhinovirus 14 and HCV
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
Ulcerative colitis (UC) is one of the two types of inflammatory bowel disease (IBD), with the other type being Crohn's disease. It is a long-term condition that results in inflammation and ulcers of the colon and rectum. The primary sympto ...
(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 mRNA 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).
Sensors
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. Furthermore, DNAzymes have been used in combination of aptamers and nucleic acid bioreceptors for the development of a multiplex bioassay.
Asymmetric synthesis
Chirality
Chirality () is a property of asymmetry important in several branches of science. The word ''chirality'' is derived from the Greek (''kheir''), "hand", a familiar chiral object.
An object or a system is ''chiral'' if it is distinguishable fro ...
is another property that a DNAzyme can exploit. DNA occurs 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
Cyclopentadiene is an organic compound with the chemical formula, formula C5H6. It is often abbreviated CpH because the cyclopentadienyl anion is abbreviated Cp−.
This colorless liquid has a strong and unpleasant odor. At room temperature, ...
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 DNA sequence.
Bioconjugation with hGQ DNAzyme
The hemin/G-Quadruplex DNAzyme consists of G-Quadruplex forming DNA that can bind the co-factor hemin (a.k.a. Fe(III)Protoporphyrin IX), forming a complex that can perform certain oxidation reaction in the presence of hydrogen peroxide. This DNAzyme can oxidize small molecules, such as dopamine and adenosine triphosphate, but can also be used for the modification of peptides and proteins by attaching small molecules.
Other uses
Other uses of DNA in chemistry are in
DNA-templated synthesis, Enantioselective catalysis,
DNA nanowires and
DNA computing
DNA computing is an emerging branch of unconventional computing which uses DNA, biochemistry, and molecular biology hardware, instead of the traditional electronic computing. Research and development in this area concerns theory, experiments, a ...
.
See also
*
*
*
References
External links
*
{{Nucleic acids
DNA
Enzymes
Catalysts
Chemical kinetics
Biomolecules