DNAzymes are catalytically active single-stranded DNAs that fold into metal-ion-assisted architectures to mediate diverse reactions. DNAzymes are artificially synthesized single-stranded DNA molecules obtained through in vitro selection. By folding into defined and often metal-ion-assisted three-dimensional structures, they form enzyme-like active centers capable of catalyzing diverse chemical reactions. Their catalytic scope extends beyond nucleic acid cleavage to include ligation, chemical modification and other organic transformations.

Deoxyribozymes, DNA enzymes or simply DNAzymes are single-stranded oligo-deoxyribonucleotide molecules that, like proteins and ribozymes, possess the ability to perform catalysis. Although DNAzymes have not yet been found in living organisms, they have been isolated in the laboratory through in vitro selection. The selected DNAzyme sequences have the ability to catalyze a broad range of chemical reactions, utilizing DNA, RNA, peptides or small organic compounds as substrates.

DNAzymes are single-stranded DNA molecules with catalytic function, which specifically cleave RNA under the action of metal ions. DNAzymes comprise two domains, one catalytic domain, and two substrate-binding domains. It combines with RNA substrate through Watson-Crick base pairing, catalyzes specific cleavage of RNA under the action of metal ions, and forms 2´, 3´—cyclic phosphate and 5´—hydroxy terminal. In addition to catalyzing RNA cleavage, DNAzymes also catalyze a series of chemical reactions, including DNA binding, DNA cleavage, RNA binding, and DNA phosphorylation.

Since most DNA molecules in nature are double stranded, they are unlikely to be catalytically active. Single-stranded DNA, on the other hand, can form tertiary structures, allowing molecular recognition and catalysis.[5] Extensive efforts have also been made in searching for DNA sequences for catalyzing other reactions, and only a few representative examples are listed here. An early example is the oxidative DNA cleavage reaction by a DNAzyme with a triplex structure, requiring both Cu2+ and ascorbate. Later, a Zn2+-dependent DNAzyme was selected, which catalyzes the DNA cleavage through hydrolysis. These and other DNAzymes illustrate that single-stranded DNA molecules can act as catalysts in a variety of biochemical reactions.[5]

Many catalytically active DNA sequences (deoxyribozymes, also called DNAzymes) have been identified in the lab by in vitro selection, in which many random DNA sequences are evaluated in parallel to find those rare sequences that have a desired functional ability.[10] Mg2+-dependent DNAzymes contain a three-way junction comprising a catalytic core flanked by two binding arms. The catalytic core is responsible for the cleavage of RNA substrates, whereas the binding arms are used to hybridize to substrates through Watson–Crick base pairing. The sequences of the binding arms define substrate recognition and allow sequence-specific RNA cleavage.[10]

Deoxyribozymes (DNAzymes) are short (15–40 nt) single-stranded DNA molecules that catalyze chemical reactions. These catalytic DNAs are not found in nature, rather they are selected from a random oligonucleotide library and enriched for a desired activity, allowing for the precise control of both the desired reaction type and condition. DNAzymes generally consist of two substrate binding arms flanking a central catalytic core. The flanking binding arms can be altered both in length and in sequence identity, allowing high substrate specificity and limiting off-target effects.

Here, we present PECAN, paired-end cutting assisted by DNAzymes (DNA enzymes or deoxyribozymes), which enables mass production of ssDNA of arbitrary sequence (up to 7000 nucleotides, or nt) with single-base precision. At the core of PECAN technique are two newly identified classes of DNAzymes, each robustly self-hydrolyzing with minimal sequence requirement up- or down-stream of its cleavage site. Our work reveals the versatility of catalytic DNA in triggering and processing biochemical reactions.

Two modified 2′-deoxynucleoside 5′-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions.[1] The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner.[1] DNAzymes have been selected that are capable of accelerating a surprising variety of chemical reactions such as RNA cleavage, DNA cleavage, DNA ligation, DNA phosphorylation, porphyrin metallation, DNA capping, DNA depurination and the Diels–Alder reaction.[1]

Deoxyribozymes are DNA molecules with catalytic activity. Their RNA analogues, ribozymes, participate in fundamental reactions of modern biochemistry such as viral RNA self-processing, RNA splicing, and translation of RNA into protein. For deoxyribozymes, the substrates have almost always been nucleic acids themselves, and a number of recent reviews have addressed the scope of DNA's catalytic activity using such substrates. This value of kcat/Km is 1–2 orders of magnitude higher than for the naturally occurring hammerhead or hairpin RNA-cleaving ribozymes, further demonstrating that DNA has no inherent catalytic inferiority relative to RNA.

The discovery of deoxyribozyme (DNAzymes) further broadened the understanding of the catalytic function of nucleic acids. In this review, we propose the concept of functional nucleic acid enzymes (FNAzymes). FNAzymes are nucleic acids or nucleic acid complexes with special structure and catalytic functions. Then FNAzymes are divided into four groups based on the components that make them up: ribozymes, DNAzymes, modified FNAzymes, and functional nucleic acid nanozymes (FNA nanozymes).

Deoxyribozymes or DNAzymes are single-stranded catalytic DNA molecules that are obtained by combinatorial in vitro selection methods. Catalytic DNA consists either of: (1) supramolecular hybrids made of metal complexes and double-helical DNA molecules or (2) of single-stranded DNA sequences (coined DNA enzymes, deoxyribozymes, or DNAzymes) obtained by in vitro selection. DNAzymes catalyzing a broad array of chemical transformations have been isolated.

Many experiments have shown that DNA can be a catalyst (deoxyribozyme). The key is to consider single-stranded DNA. In its single-stranded form, DNA is similar to RNA with regard to many biochemical properties, particularly the ability to fold into secondary structures and intricate three-dimensional conformations. This structure reveals several important facets of catalysis by DNA, including the fundamental confirmation that deoxyribozymes can have well-defined tertiary structures that are responsible for their catalysis.

A new DNA enzyme, the "Bipartite DNAzyme", suitable for the sequence-specific cleavage of RNA, was obtained from a random DNA library by in vitro selection.[3] The bipartite DNAzyme contains a catalytic domain and substrate-recognition domains; it cleaves its RNA substrate at a specific site defined by complementary base-pairing interactions, demonstrating that DNA enzymes can perform sequence-specific biochemical reactions on RNA.[3]

DNAzymes | Catalytic DNA molecules that mediate cleavage, ligation, phosphorylation, base excision, capping, and oxidation reactions on nucleic acid or small-molecule substrates | DNAzyme (RNA-cleaving DNAzyme) | The DNAzyme is a small catalytic DNA motif with a conserved core and two substrate-binding arms. It cleaves RNA at a specific ribonucleotide site, producing 2′,3′-cyclic phosphate and 5′-hydroxyl termini. Catalysis requires divalent metal ions (most efficiently Pb2+, but also Mg2+, Zn2+, or Mn2+) and the sequence specificity is programmable via Watson–Crick base pairing.

DNAzymes, also known as deoxyribozymes or DNA enzymes, refer to single-stranded DNA molecules with catalytic capabilities. DNAzymes are generated de novo by in vitro selection—a powerful and yet simple technique that has been routinely used to isolate extremely rare DNA or RNA sequences with a function of interest (e.g. ligand-binding or catalysis) from an extraordinarily large population of single-stranded DNA or RNA molecules.

Since most DNA molecules in nature are double stranded, they are unlikely to be catalytically active. Single-stranded DNA, on the other hand, can form tertiary structures, allowing molecular recognition and catalysis. In the early 1990s, single-stranded DNAs with specific binding activity (aptamers) have been obtained through in vitro selection, followed by the discovery of catalytic DNAs (DNAzymes, deoxyribozymes).

DNA‐based enzymes, also known as deoxyribozymes or DNAzymes, are single‐stranded DNA molecules with catalytic activity. DNAzymes do not exist in nature but can be isolated from random‐sequence DNA pools using in vitro selection. Furthermore, the sequence of the substrate binding arms of RNA-cleaving DNAzymes determines the unique RNA substrate sequence, thus allowing for highly sequence-specific recognition and cleavage.

To ensure sequence-specificity, such RNase A mimics have been conjugated to oligonucleotides, PNAs or peptides that are used to deliver elements of sequence specificity.[4] In terms of sequence specificity, Dz7-38-32t showed >20-fold selectivity for the target sequence compared to targets which differed in terms of the nucleobase located 5′ with respect to the cleavage site, illustrating that engineered DNAzymes can catalyze RNA cleavage reactions with high sequence selectivity.[4]

However, the available experimental evidence suggests that DNA and RNA actually have comparable catalytic efficiencies, at least where direct comparisons have been made. The rate enhancements for deoxyribozymes as listed in Table 1 support this conclusion, because analogous rate enhancements for ribozymes are in the same range. Deoxyribozymes therefore represent genuine catalytic nucleic acids, despite lacking the 2′-hydroxyl group of RNA.

Herein, we have probed the fundamental requirements for DNA catalysis, and shown that a simpler alphabet comprised of fewer than the standard four types of deoxyribonucleotides can support appreciable catalytic activity. More importantly, we have shown that nucleic acid catalysis is possible with an even simpler alphabet comprised of only guanosine and cytidine deoxyribonucleotides. However, we have demonstrated that a specific sequence composed of just seven guanosine and six cytidine nucleotides can form the catalytic core of a functional RNA-cleaving DNAzyme.

DNAzymes (deoxyribozymes) are single-stranded DNA molecules endowed with catalytic activity, obtained by in vitro selection. This review examines the functional information gathered on 8-17, in the light of the available crystal structures, pointing out the congruences and possible inconsistencies between the functional and structural data.

Single-stranded DNA molecules have the capacity to adopt catalytically active structures known as DNAzymes, although the fundamental limits of this ability have not been determined. However, we have demonstrated that a specific sequence composed of just seven guanosine and six cytidine nucleotides can form the catalytic core of a functional RNA-cleaving DNAzyme.

Nevertheless, many experiments have shown that DNA can be a catalyst (deoxyribozyme). Several pertinent reviews have addressed the field of deoxyribozymes, including both general overviews and the relationship of DNA catalysis to other applications of DNA. This review describes the advantages of DNA as a catalyst and highlights recent advances in the scope, applications, and structural investigation of deoxyribozymes.

The RNA cleaving DNAzymes have very similar catalytic properties to ribozymes isolated from plant virusoids and viroids. They produce 5′-OH and 2′,3′-cyclic phosphate termini at the cleavage site and targeting is determined by Watson-Crick base pairing. An important distinction relative to the delivery of ribozymes versus DNAzymes is that ribozymes can be delivered to tissues or cells as genes to be expressed or as synthetic RNA molecules, whereas DNAzymes only can be delivered as synthetic oligonucleotides.

DNAzymes, a class of single-stranded catalytic DNA with good stability, high catalytic activity, and easy synthesis, functionalization and modification properties, have garnered significant interest in the realm of biosensing and bioimaging. Their integration with fluorescent dyes or chemiluminescent moieties has led to remarkable bioimaging outcomes, while DNAzyme-based biosensors have demonstrated robust sensitivity and selectivity in detecting metal ions, nucleic acids, proteins, enzyme activities, exosomes, bacteria and microorganisms.

DNAzymes are single-stranded DNA molecules with catalytic function, which specifically cleave RNA under the action of metal ions. DNAzymes comprise two domains, one catalytic domain, and two substrate-binding domains. It combines with RNA substrate through Watson–Crick base pairing, catalyzes specific cleavage of RNA under the action of metal ions, and forms 2',3'-cyclic phosphate and 5'-hydroxy terminal.

RNA-cleaving DNAzymes, which are single-stranded catalytic DNA, have attracted considerable attention in bioanalysis and biomedical applications because of their high stability, high catalytic activity, easy synthesis, easy functionalization, and modification. This review systematically summarizes the applications of RNA-cleaving DNAzymes in recent years, explaining the uniqueness and superiority of RNA-cleaving DNAzymes in biosensing and gene therapy.

Therefore, it is believed that RNA-cleavage mediated by both Dz8-17 and Dz10-23 follows a mechanism that is similar to that of ribozymes such as the hammerhead ribozyme. DNA catalysis has been extended beyond RNA cleavage to numerous other reactions, demonstrating that DNAzymes are versatile catalytic single-stranded DNA molecules whose activity and sequence specificity can be tailored by in vitro selection.

RNA-cleaving deoxyribozymes (DNAzymes) are synthetic single-stranded DNA-based catalytic molecules that can be engineered to bind to and cleave target mRNA at predetermined sites. DNAzymes are synthetic single-stranded enzymatic DNA molecules that bind to their target mRNA via Watson–Crick base pairing and cleave a specific interbase junction in the mRNA by a deesterification reaction.

DNAzymes (Dzs) are single-stranded DNA catalysts that specifically cleave the mRNA of targeted genes. DNAzymes, first synthesized experimentally in 1994 by Breaker and Joyce, are single-stranded DNA sequences with catalytic activity, often designed so that their binding arms recognize a target sequence and their catalytic core performs site-specific cleavage.

Deoxyribozymes, also called DNAzymes, are single-stranded DNA molecules with catalytic activity. 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.[9] A DNA molecule with sequence 5'-GGAGAACGCGAGGCAAGGCTGGGAGAAATGTGGATCACGATT-3' acts as a deoxyribozyme that uses light to repair a thymine dimer, using serotonin as cofactor, showing that DNAzymes can catalyze other biochemical reactions such as photorepair of DNA lesions.[9]

In the early 1980s, RNA molecules that can catalyze enzymatic reactions were discovered and named ribozymes. This discovery was followed by demonstrations in the 1990s that DNA also can act as enzymes, termed deoxyribozymes or DNAzymes.[11] These DNAzymes are short DNA sequences that fold into specific three-dimensional structures enabling them to catalyze reactions such as site-specific cleavage of RNA, highlighting that DNA can serve as a catalytic, sequence-dependent biomolecule.[11]

Although natural sources of enzymes are limited to protein and RNA, some artificial DNAs exhibit catalytic activities. Representative functions of such DNAs, i.e. DNAzymes, are cleavage and ligation of nucleic acids. A minimal RNA-cleaving DNAzyme was identified that retains catalytic activity despite having a very short core sequence, highlighting that single-stranded DNA can fold into compact structures that support sequence-specific catalysis.

In vitro selection from random sequence libraries has permitted the isolation and characterization of a variety of DNAzymes, including those for RNA cleavage. The hammerhead and hairpin ribozymes have been modified to cleave any substrate RNA sequence site-specifically, and similar approaches have yielded DNAzymes that cleave RNA in a sequence-specific manner.[2] These studies demonstrate that DNA enzymes can be tailored to recognize and catalytically cleave particular RNA sequences based on Watson–Crick complementarity, confirming their ability to perform sequence-specific biochemical reactions.[2]

Single-stranded DNA in nature can form complex tertiary structures similar to RNA, allowing molecular recognition and catalysis. Artificially selected deoxyribozymes are typically single-stranded and rely on defined sequences to fold into catalytic motifs, enabling sequence-specific reactions such as RNA cleavage and ligation.[5] This reflects a broader principle that DNA enzymes are catalytic, single-stranded DNA molecules whose specific nucleotide sequences determine both their three-dimensional structure and their biochemical reaction specificity.