Deoxyribozyme
Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are 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).[1] However, in contrast to the abundance of protein enzymes in biological systems and the discovery of biological ribozymes in the 1980s,[2][3] there is only little evidence for naturally occurring deoxyribozymes.[4][5] 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
In addition to the inherent inferiority of DNA catalytic activity, the apparent lack of naturally occurring deoxyribozymes may also be due to the primarily
In 2021, the DNAmoreDB database for cataloguing known deoxyribozymes was released.[7]
Types
Ribonucleases
The most abundant class of deoxyribozymes are
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 pairs.The first known deoxyribozyme was a ribonuclease, discovered in 1994 by
A DNA molecule with sequence 5'-GGAGAACGCGAGGCAAGGCTGGGAGAAATGTGGATCACGATT-3' acts as a deoxyribozyme that uses light to repair a
RNA ligases
Of particular interest are DNA
Other reactions
Many other deoxyribozymes have since been developed that catalyze DNA phosphorylation, DNA
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.[25][26] in vitro selection utilizes a "pool" of a large number of random 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.[26]
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 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 tag with a streptavidin affinity column.[25][26] 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.[26] 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,[8] 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.[26] The dynamics of the pool can be described through mathematical modeling,[27] 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
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.
"True" catalysis?
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.[6] Furthermore, many endogenous enzymes (both proteins and ribozymes) also exhibit single-turnover behavior,[6] and so the exclusion of deoxyribozymes from the rank of "catalyst" simply because it does not feature multiple-turnover behavior seems unjustified.
Applications
Although RNA enzymes were discovered before DNA enzymes, the latter have some distinct advantages. DNA is more
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.[39] 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.[40] 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.[41] 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).[15]
Sensors
DNAzymes have found practical use in metal biosensors.[42][43] A DNAzyme based biosensor for lead ion was used to detect lead ion in water in St. Paul Public Schools in Minnesota.[44] Furthermore, DNAzymes have been used in combination of aptamers and nucleic acid bioreceptors for the development of a multiplex bioassay.[45]
Asymmetric synthesis
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.[47] This DNAzyme can oxidize small molecules, such as dopamine and adenosine triphosphate,[48] but can also be used for the modification of peptides[49] and proteins[50][51] by attaching small molecules.
Other uses
Other uses of DNA in chemistry are in
See also
- Aptamer – Oligonucleotide or peptide molecules that bind specific targets
- Ribozyme – Type of RNA molecules
- Systematic evolution of ligands by exponential enrichment (SELEX) – Technique for producing oligonucleotides that specifically bind to a target
References
- ^ S2CID 1918660.
- S2CID 14787080.
- ^
Guerrier-Takada C, Gardiner K, Marsh T, Pace N, Altman S (December 1983). "The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme". Cell. 35 (3 Pt 2): 849–857. S2CID 39111511.
- PMID 32280846.
- PMID 25237854.
- ^ PMID 15455136.
- PMID 33053178.
- ^ PMID 16286368.
- ^ PMID 9383394.
- PMID 20407665.
- S2CID 8546430.
- ISSN 1521-3773.
- PMID 9113977.
- ^
Cruz RP, Withers JB, Li Y (January 2004). "Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme". Chemistry & Biology. 11 (1): 57–67. PMID 15112995.
- ^ PMID 22352843.
- PMID 37761982.
- ISSN 0002-7863.
- PMID 23531046.
- PMID 25918425.
- S2CID 4461523.
- ^ Borman S. "After Two Decades Of Trying, Scientists Report First Crystal Structure Of A DNAzyme | January 11, 2016 Issue - Vol. 94 Issue 2 | Chemical & Engineering News". cen.acs.org. Retrieved 2017-02-04.
- S2CID 53010843.
- PMID 14691255.
- ^
Chinnapen DJ, Sen D (January 2004). "A deoxyribozyme that harnesses light to repair thymine dimers in DNA". Proceedings of the National Academy of Sciences of the United States of America. 101 (1): 65–69. PMID 14691255.
- ^ a b c
Joyce GF (2004). "Directed evolution of nucleic acid enzymes". Annual Review of Biochemistry. 73 (1): 791–836. PMID 15189159.
- ^ a b c d e f g
Silverman SK (August 2008). "Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects". Chemical Communications (30): 3467–3485. S2CID 9824687.
- ^
Spill F, Weinstein ZB, Irani Shemirani A, Ho N, Desai D, Zaman MH (October 2016). "Controlling uncertainty in aptamer selection". Proceedings of the National Academy of Sciences of the United States of America. 113 (43): 12076–12081. PMID 27790993.
- PMID 26091540.
- ^
Paul N, Springsteen G, Joyce GF (March 2006). "Conversion of a ribozyme to a deoxyribozyme through in vitro evolution". Chemistry & Biology. 13 (3): 329–338. PMID 16638538.
- ^ Kumar B, Asha K, Chauhan SP (2013-10-07). "DNAzyme Mediated Post-transcriptional Gene Silencing: A Novel Therapeutic Approach".
- S2CID 45686564.
- S2CID 23234776.
- PMID 23971908.
- PMID 28039102.
- PMID 29322273.
- ^ PMID 30577479.
- PMID 14530446.
- PMID 18559927.
- PMID 25981191.
- ^ "Efficacy, Pharmacokinetics, Tolerability, Safety of SB012 Intrarectally Applied in Active Ulcerative Colitis Patients (SECURE)". ClinicalTrials.gov. Retrieved May 27, 2016.
- ^ "Efficacy, Safety, Tolerability, Pharmacokinetics and Pharmacodynamics Study of the Topical Formulation SB011 Applied to Lesional Skin in Patients With Atopic Eczema". ClinicalTrail.gov. Retrieved May 27, 2016.
- .
- S2CID 5201672.
- ^ "Lead in Water: St. Paul Schools Delayed Fixes". ABC 6 NEWS. Archived from the original on 2021-12-08. Retrieved 2017-02-04.
- PMID 34184101.
- S2CID 2317268.
- PMID 9751647.
- PMID 26652164.
- S2CID 238732951.
- PMID 32909740.
- PMID 31037152.
- PMID 22210342.
- doi:10.1016/j.molcatb.2004.01.016. Archived from the original(PDF) on 2005-10-28.
External links
- Deoxyribozyme at the U.S. National Library of Medicine Medical Subject Headings (MeSH)