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Methods for modifying intact peptides are useful but can be unselective with regard to amino acid position and sequence context. In this work, we used in vitro selection to identify DNAzymes that acylate a Lys residue of a short peptide in sequence-dependent fashion. The DNAzymes do not acylate Lys when placed at other residues in the peptide, and the acylation activity depends on the Lys sequence context. A high acylation yield is observed on the preparative nanomole scale. These findings are promising for further development of DNAzymes for broader application to top-down Lys acylation of peptide and protein substrates., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis., (© 2024 Wiley-VCH GmbH.)

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Cleavage of biological mRNA by DNAzymes (Dz) has been proposed as a variation of oligonucleotide gene therapy (OGT). The design of Dz-based OGT agents includes computational prediction of two RNA-binding arms with low affinity (melting temperatures (T m ) close to the reaction temperature of 37 °C) to avoid product inhibition and maintain high specificity. However, RNA cleavage might be limited by the RNA binding step especially if the RNA is folded in secondary structures. This calls for the need for two high-affinity RNA-binding arms. In this study, we optimized 10-23 Dz-based OGT agents for cleavage of three RNA targets with different folding energies under multiple turnover conditions in 2 mM Mg 2+ at 37 °C. Unexpectedly, one optimized Dz had each RNA-binding arm with a T m ≥60 °C, without suffering from product inhibition or low selectivity. This phenomenon was explained by the folding of the RNA cleavage products into stable secondary structures. This result suggests that Dz with long (high affinity) RNA-binding arms should not be excluded from the candidate pool for OGT agents. Rather, analysis of the cleavage products' folding should be included in Dz selection algorithms. The Dz optimization workflow should include testing with folded rather than linear RNA substrates., (© 2023 Wiley-VCH GmbH.)

Trypanosoma brucei is the causal infectious agent of African trypanosomiasis in humans and Nagana in livestock. Both diseases are currently treated with a small number of chemotherapeutics, which are hampered by a variety of limitations reaching from efficacy and toxicity complications to drug-resistance problems. Here, we explore the forward design of a new class of synthetic trypanocides based on nanostructured, core-shell DNA-lipid particles. In aqueous solution, the particles self-assemble into micelle-type structures consisting of a solvent-exposed, hydrophilic DNA shell and a hydrophobic lipid core. DNA-lipid nanoparticles have membrane-adhesive qualities and can permeabilize lipid membranes. We report the synthesis of DNA-cholesterol nanoparticles, which specifically subvert the membrane integrity of the T. brucei lysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA shell with a spliced leader (SL)-RNA-specific DNAzyme, we target a second trypanosome-specific pathway (dual-target approach). The DNAzyme provides a backup to counteract the recovery of compromised parasites, which reduces the risk of developing drug resistance., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Chemical gradient sensing behavior of catalytically active colloids and enzymes is an area of immense interest owing to their importance in understanding fundamental spatiotemporal complexity patterns in living systems and designing dynamic materials. Herein, we have shown the peroxidase activity of DNAzyme (G-quadruplex-hemin complex tagged in a micron-sized glass bead) can be modulated by metal ions and metal ion-binding oligonucleotides. Next we demonstrated both experimentally and theoretically, that the localization and product formation ability of the DNAzyme-containing particle remains biased to the more catalytically active zone where the concentration of metal ion (Hg 2+ ) inhibitor is low. Interestingly, this biased localization can be broken by introduction of Hg 2+ binding oligonucleotide in the system. Additionally, a macroscopically asymmetric catalytic product distributed zone has been achieved with this process, showing the possibility of regulation in autonomous spatially controlled chemical processes. This demonstration of autonomous modulation of the localization pattern and spatially specific enhanced product forming ability of DNAzymes will further enable the design of responsive nucleic acid-based motile materials and surfaces., (© 2022 Wiley-VCH GmbH.)

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The conditional control of protein function in response to the physiological change of cells is of great interest for studying protein function in biological settings and developing protein therapeutics. We report herein that catalase (CAT) DNAzyme can potentiate the generation of reactive oxygen species (ROS) in living cells by knocking down catalase expression, which could further activate a reactive oxygen species (ROS)-responsive pro-protein, RNase A-NBC, in situ. Using an optimized lipid nanoparticle delivery system to simultaneously introduce CAT DNAzyme and RNase A-NBC into cells, we show that the pro-protein, RNase A-NBC, could be activated in a significantly enhanced manner to prohibit tumor cell growth in different types of cancer cells. We believe the methodology of regulating pro-protein activity using DNAzyme biocatalysis to differentiate intracellular environment could further be extended to other functional proteins, and even fundamental investigations in living systems to develop pro-protein therapeutics., (© 2021 Wiley-VCH GmbH.)

Functional nucleic acids (FNAs), including naturally occurring ribozymes and riboswitches as well as artificially created DNAzymes and aptamers, have been popular molecular toolboxes for diverse applications. Given the high chemical stability of nucleic acids and their ability to fold into diverse sequence-dependent structures, FNAs are suggested to be highly functional under unusual reaction conditions. This review will examine the progress of research on FNAs under conditions of low pH, high temperature, freezing conditions, and the inclusion of organic solvents and denaturants that are known to disrupt nucleic acid structures. The FNA species to be discussed include ribozymes, riboswitches, G-quadruplex-based peroxidase mimicking DNAzymes, RNA-cleaving DNAzymes, and aptamers. Research within this space has not only revealed the hidden talents of FNAs but has also laid important groundwork for pursuing these intriguing functional macromolecules for unique applications., (© 2021 Wiley-VCH GmbH.)

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment., (© 2021 Wiley-VCH GmbH.)

Since 1994, deoxyribozymes or DNAzymes have been in vitro selected to catalyze various types of reactions. Metal ions play a critical role in DNAzyme catalysis, and Zn 2+ is a very important one among them. Zn 2+ has good biocompatibility and can be used for intracellular applications. Chemically, Zn 2+ is a Lewis acid and it can bind to both the phosphate backbone and the nucleobases of DNA. Zn 2+ undergoes hydrolysis even at neutral pH, and the partially hydrolyzed polynuclear complexes can affect the interactions with DNA. These features have made Zn 2+ a unique cofactor for DNAzyme reactions. This review summarizes Zn 2+ -dependent DNAzymes with an emphasis on RNA-/DNA-cleaving reactions. A key feature is the sharp Zn 2+ concentration and pH-dependent activity for many of the DNAzymes. The applications of these DNAzymes as biosensors for Zn 2+ , as therapeutic agents to cleave intracellular RNA, and as chemical biology tools to manipulate DNA are discussed. Future studies can focus on the selection of new DNAzymes with improved performance and detailed biochemical characterizations to understand the role of Zn 2+ , which can facilitate practical applications of Zn 2+ -dependent DNAzymes., (© 2020 Wiley-VCH GmbH.)

The regulation of DNAzyme activity is an important problem for its in vivo applications. We achieved photochemical regulation of DNAzyme activity by using reversible DNA photo-crosslinking of 3-cyanovinylcarbazole ( CNV K). The ODN containing CNV K photo-crosslinked to a pyrimidine base in the complementary strand after a few seconds of photoirradiation, and its photoadduct was split by photoirradiation of another wavelength. The activity of photo-crosslinked DNAzyme with CNV K was completely inhibited (OFF state). In contrast, after 312 nm irradiation, DNAzyme activity was recovered upon addition of a substrate strand (ON state). In addition, the photo-crosslinked DNAzyme is prone to enzymatic digestion by exonuclease. This photochemical OFF to ON switching with reversible DNA photo-crosslinking was regulated at the desired time and position; therefore, it might be possible to use it for in vivo application., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We herein report a DNAzyme named T30695 (sequence: (G 3 T) 4 ) that can catalyze Zn 2+ insertion into three different porphyrins in the presence of Pb 2+ as a cofactor. Meanwhile, T30695 with Pb 2+ alone was found to cause a shift in both the fluorescence and UV-vis spectra of protoporphyrin IX (PPIX), thus suggesting that metalation of Pb 2+ was also achieved at room temperature. From kinetic measurements, the reaction required two Pb 2+ ions; this is consistent with one being a cofactor and the other being a substrate. No previous reports inserted Pb 2+ into porphyrins by using DNAzymes or protein-based enzymes. This reaction was most significantly inhibited in the presence of K + followed by Na + and Li + , suggesting the importance of the Pb 2+ -stabilized G-quadruplex. When Pb 2+ is inserted into PPIX, its emission blue shifts from 635 to 590 nm, thus allowing simple ratiometric fluorescent sensing with a detection limit of 1.2 nM Pb 2+ ., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology-controlled or enabled molecular devices. This article will begin by summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts to couple functional nucleic acids with rolling circle amplification for ultra-sensitive biosensing and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery will be recapitulated., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb 2+ for its RNA cleavage activity. Three years later, the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb 2+ , although other divalent metals can work as well. GR5 and 17E have the same substrate sequence, and their catalytic loops in the enzyme strands also have a few similar and conserved nucleotides. Considering these, we hypothesized that 17E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17E. By using the activity ratio in the presence of Pb 2+ and Mg 2+ for defining these two DNAzymes, the critical nucleotide was identified to be T 12 in 17E for metal specificity. In addition, G 9 in GR5 is a position not found in most 17E or 8-17 DNAzymes, and G 9 needs to be added to rescue GR5 activity if T 12 becomes a cytosine. This study highlights the links between these two classic and widely used DNAzymes, and offers new insight into the sequence-activity relationship related to metal selectivity., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The discovery of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that natural genetic polymers are not unique in their ability to function as enzymes. However, all known examples of in vitro selected XNA enzymes function with lower activity than their natural counterparts, suggesting that XNAs might be limited in their ability to fold into structures with high catalytic activity. To explore this problem, we evaluated the catalytic potential of FANAzyme 12-7, an RNA-cleaving catalyst composed entirely of 2'-fluoroarabino nucleic acid (FANA) that was evolved to cleave RNA at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Here, we show that this activity extends to chimeric DNA substrates that contain a central riboguanosine (riboG) residue at the cleavage site. Surprisingly, FANAzyme 12-7 rivals known DNAzymes that were previously evolved to cleave chimeric DNA substrates under physiological conditions. These data provide convincing evidence that FANAzyme 12-7 maintains the catalytic potential of equivalent DNAzymes, which has important implications for the evolution of XNA catalysts and their contributions to future applications in synthetic biology., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It has been proposed that Mg 2+ and Fe 2+ are very similar in interacting with ribozymes and some protein-based enzymes, but their activities with DNAzymes have yet to be studied. Here, the activity of Fe 2+ as cofactor for a few RNA-cleaving DNAzymes is investigated. 17E is a well-studied DNAzyme that is active in the presence of many different divalent metal ions; it is highly active with Fe 2+ with an apparent K d of 29.7±2.3 μm and a k obs of 1.12±0.11 min -1 in the presence of 1 mm Fe 2+ at pH 7.5. Fe 2+ has 21-fold higher activity than Mg 2+ . Six different DNAzymes are then tested, and only the DNAzymes active with Mg 2+ (17E, 8-17, and E5) are active with Fe 2+ . Fe 2+ has 25 and one- to twofold higher activity than Mg 2+ for the 8-17 and E5 DNAzymes, respectively. In pH>7 buffer and in presence of air, 1 mm Fe 2+ results in a nonspecific degradation of the DNA strand due to reactive oxygen species (ROS). Cleavage reactions in anoxic environment and antioxidant ascorbate can be used to overcome the effect of oxidation. The findings provide insights for potential DNAzyme catalysis in the early Earth, and they further support the similarity between Mg 2+ and Fe 2+ in enzyme catalysis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sodium is one of the most common metal ions in biology; however, DNA-based sodium probes have only been reported recently. A Na + -specific RNA-cleaving DNAzyme named NaA43 is active with Na + alone. In this work, we were using Co(NH 3 ) 6 3+ as the intended metal cofactor for in vitro selection, but obtained a mutant of the NaA43 DNAzyme. The mutant was named NaH1, and differs from NaA43 by only two nucleotides. NaA43 has an optimal pH of 7.0, whereas the optimal pH for NaH1 is 6.0. This difference might be due to our selection having been performed at pH 6.0. NaH1 also displays an excellent selectivity for sodium relative to other competing monovalent ions, as well as a fast catalytic rate of (0.11±0.01) min -1 with 50 mm Na + . At low Na + concentrations, the selected DNAzyme exhibited a higher cleavage rate than NaA43 and thus a tighter apparent K d of (12.0±1.6) mm Na + . Furthermore, the NaH1 DNAzyme was engineered into a fluorescent Na + biosensor by attaching a fluorophore/quencher pair to the DNAzyme with a detection limit of 223 μm Na + . Preliminary work on detection of Na + in serum was demonstrated as well. This study provides a useful mutant that works in a slightly acidic environment, which might be useful for sensing Na + in acidic in vivo environments., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently, a few RNA-cleaving DNAzymes have been isolated with excellent specificity for Na + , and some of them contain a Na + -binding aptamer. This metal recognition mechanism is different from that of most previously reported DNAzymes. When using 2-aminopurine (2AP) as a probe, interesting local folding induced by Na + was recently observed. In this work, FRET was used to probe the global folding of the Ce13d DNAzyme; one of the Na + -specific DNAzymes. FRET pairs were at different locations, which yielded a total of five constructs to probe the three-way junction structure with a large loop. With endlabeled DNAzymes, the global structure appears to be quite rigid with little folding upon adding up to 200 mm monovalent metal ions, although some minor differences were observed between Li + , Na + , and K + . This lack of significant conformational change is also consistent with circular dichroism spectroscopy data. The loop was then labeled with an internal tetramethylrhodamine fluorophore at the G14 position, and its cleavage activity was partially retained. A clear Na + -dependent folding was observed with spectral crossover. From a biosensing standpoint, global folding based sensors are unlikely to work due to the overall rigid structure of the DNAzyme. Therefore, the best way to use this DNAzyme to discriminate Na + from K + is based on cleavage activity, followed by probing local folding, whereas global folding is the least effective for metal discrimination., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The EtNa DNAzyme was isolated during the isopropanol precipitation step of an in vitro selection effort. Although inactive with the intended cofactor, its RNA cleavage activity was observed under a few conditions. With Na + , EtNa was highly active in ∼50 % ethanol, whereas in water, it was highly active with Ca 2+ . In this work, we showed that the EtNa DNAzyme was accelerated by freezing in water in the presence of Na + . The apparent K d value reached 6.2 mm Na + under the frozen condition, over 20 times tighter than that in water at room temperature. With 10 mm Na + , EtNa had a cleavage rate of 0.12 h -1 after freezing at -20 °C. This effect was unique to EtNa, as all other tested DNAzymes were inhibited by freezing except for the Na + -specific NaA43. Freezing also inhibited EtNa if Ca 2+ was used. We attributed this to the concentrations of EtNa and Na + in the micropockets between ice crystals, but divalent metals might misfold DNA. Overall, we have systematically studied the effect of freezing on the RNA-cleavage activity of DNAzymes. The DNAzyme sequence and the metal ion species are both crucial to determine the effect of freezing., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Functional DNA includes aptamers and DNAzymes, and metal ions are often important for achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA-based biosensors. In this work, the effect of ethanol on the activity of the EtNa DNAzyme was studied for Ca 2+ detection. With 30 % ethanol, the sensor has a detection limit of 1.4 μm Ca 2+ , which is a 16-fold improvement relative to that in water. This EtNa DNAzyme is unique because other tested DNAzymes are all inhibited by 50 % ethanol. Finally, by using the EtNa DNAzyme as a scaffold, the adenosine monophosphate (AMP) aptamer was inserted to construct an aptazyme, which allowed the measurement of AMP in ethanol. In summary, this study has reported the most sensitive DNA-based sensor for Ca 2+ , and its sensitivity and selectivity can approach those of proteins or small-molecule ligands. This work also provides a way to measure aptamer binding in organic solvents., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Our view of the interaction between Na + and nucleic acids was changed by a few recently discovered Na + -specific RNA-cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na + binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140-fold, and it cannot be rescued by thiophilic Cd 2+ or Mn 2+ , whereas the activity of PS-modified Ce13d can be rescued. Na + -dependent activity assays indicate that two Na + ions bind cooperatively in EtNa, and each Na + likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na + ion in a well-defined Na + aptamer, and this Na + ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH-rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na + fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na + binding is detected by 2-aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na + mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well-defined aptamer for Na + binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na + binding., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ca 2+ is a highly important metal ion in biology and in the environment, and thus there is extensive work in developing sensors for Ca 2+ detection. Although many Ca 2+ -binding proteins are known, few nucleic acids can selectively bind Ca 2+ . DNA-based biosensors are attractive for their high stability and excellent programmability. We report a RNA-cleaving DNAzyme, EtNa, cooperatively binding two Ca 2+ ions but to only one Mg 2+ . Four DNAzymes with known Ca 2+ -dependent activity were compared, and the EtNa had the best selectivity for Ca 2+ . The EtNa is 90 times more active in Ca 2+ than in Mg 2+ . Phosphorothioate (PS) modification showed that both non-bridging oxygen atoms at the scissile phosphate contribute equally to Ca 2+ binding. The pH-rate profile suggests two concurrent deprotonation reactions. EtNa was further engineered for Ca 2+ sensing, and found to have a detection limit of 17 μm Ca 2+ and excellent selectivity. The detection of Ca 2+ in tap water was performed, and the result was comparable with that by ICP-MS. This study offers new fundamental insights into Ca 2+ binding by nucleic acids and improved metal selectivity by having multiple cooperative metal binding sites., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Some natural enzymes increase the rate of diffusion-limited reactions by facilitating substrate flow to their active sites. Inspired by this natural phenomenon, we developed a strategy for efficient substrate delivery to a deoxyribozyme (DZ) catalytic sensor. This resulted in a three- to fourfold increase in sensitivity and up to a ninefold improvement in the detection limit. The reported strategy can be used to enhance catalytic efficiency of diffusion-limited enzymes and to improve sensitivity of enzyme-based biosensors., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A previous study of two RNA-cleaving DNAzymes, NaA43 and Ce13d, revealed the possibility of a common Na(+) aptamer motif. Because Na(+) binding to DNA is a fundamental biochemical problem, the interaction between Ce13d and Na(+) was studied in detail by using sensitized Tb(3+) luminescence spectroscopy. Na(+) displaces Tb(3+) from the DNAzyme, and thus quenches the emission from Tb(3+) . The overall requirement for Na(+) binding includes the hairpin and the highly conserved 16-nucleotide loop in the enzyme strand, along with a few unpaired nucleotides in the substrate. Mutation studies indicate good correlation between Na(+) binding and cleavage activity, thus suggesting a critical role of Na(+) binding for the enzyme activity. Ce13d displayed a Kd of ∼20 mm with Na(+) (other monovalent cations: 40-60 mm). The Kd values for other metal ions are mainly due to non-specific competition. With a single nucleotide mutation, the specific Na(+) binding was lost. Another mutant improved Kd to 8 mm with Na(+) . This study has demonstrated a Na(+) aptamer with important biological implications and analytical applications. It has also defined the structural requirements for Na(+) binding and produced an improved mutant., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNA can form two types of linkage. In addition to the predominant 3'-5' linkage, 2'-5'-linked RNA is also important in biology, medicine, and prebiotic studies. Here, in vitro selection was used to isolate a DNAzyme that specifically cleaves 2'-5' RNA by using Ce(3+) as the metal cofactor, but leaves the 3'-5' counterpart intact. This Ce5 DNAzyme requires trivalent light lanthanide ions and shows a rate of 0.16 min(-1) in the presence of 10 μm Ce(3+) ; the activity decreases with heavier lanthanide ions. This is the fastest DNAzyme reported for this reaction, and it might enable applications in chemical biology. As a proof-of-concept, using this DNAzyme, the reactions between phosphorothioate-modified RNA and strongly thiophilic metals (Hg(2+) and Tl(3+) ) were studied as a function of pH., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzymes working in organic solvents are important for analytical chemistry, catalysis, and mechanistic studies. Although a few protein enzymes are highly active in organic solvents, little is known regarding nucleic acid-based enzymes. Herein, we report the first RNA-cleaving DNAzyme, named EtNa, that works optimally in concentrated organic solvents containing only monovalent Na(+). The EtNa DNAzyme has a rate of 2.0 h(-1) in 54% ethanol (with 120 mM NaCl and no divalent metal ions), and a Kd of 21 mm Na(+). It retains activity even in 72% ethanol as well as in DMSO. With 4 mm Na(+), the rate in 54% ethanol is >1000-fold higher than that in water. We also demonstrated the use of EtNa to measuring the ethanol content in alcoholic drinks. In total, this DNAzyme has three unique features: divalent metal independent activity, Na(+) selectivity among monovalent metals, and acceleration by organic solvents., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemical reactions catalyzed by DNAzymes offer a route to programmable modification of biomolecules for therapeutic purposes. To this end, we have developed a new type of catalytic DNA-based logic gates in which DNAzyme catalysis is controlled via toehold-mediated strand displacement reactions. We refer to these as DNAzyme displacement gates. The use of toeholds to guide input binding provides a favorable pathway for input recognition, and the innate catalytic activity of DNAzymes allows amplification of nanomolar input concentrations. We demonstrate detection of arbitrary input sequences by rational introduction of mismatched bases into inhibitor strands. Furthermore, we illustrate the applicability of DNAzyme displacement to compute logic functions involving multiple logic gates. This work will enable sophisticated logical control of a range of biochemical modifications, with applications in pathogen detection and autonomous theranostics., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the potential to engineer biological systems, synthetic biology is an emerging field that combines various disciplines of sciences. It encompasses combinations of DNA, RNA and protein modules for constructing desired systems and the "rewiring" of existing signalling networks. Despite recent advances, this field still lags behind in the artificial reconstruction of cellular processes, and thus demands new modules and switches to create "genetic circuits". The widely characterised noncanonical nucleic acid secondary structures, G-quadruplexes are promising candidates to be used as biological modules in synthetic biology. Structural plasticity and functional versatility are significant G-quadruplex traits for its integration into a biological system and for diverse applications in synthetic circuits., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the blink of the eye: a cascade of two deoxyribozymes was designed for rapid visual detection of bacterial 16S rRNA. The detection limit is 12.5 ng by the naked eye, with the ability to differentiate between closely related pathogenic and nonpathogenic species., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photolysis of an aryl sulfide-containing 5,6-dihydropyrimidine (1) at 350 nm produces high yields of thymidine and products resulting from trapping of a 5,6-dihydrothymidin-5-yl radical by O₂ or thiols. Thymidine is believed to result from disproportionation of the radical pair originally generated from C--S bond homolysis of 1 on the microsecond timescale, which is significantly shorter than other photochemical transformations of modified nucleotides into their native forms. Duplex DNA containing 1 is destabilized, presumably due to disruption of π-stacking. Incorporation of 1 within the binding site of the restriction endonuclease EcoRV provides a photochemical switch for turning on the enzyme's activity. In contrast, 1 is a substrate for endonuclease VIII and serves as a photochemical off switch for this base excision repair enzyme. Modification 1 also modulates the activity of the 10-23 DNAzyme, despite its incorporation into a nonduplex region. Overall, dihydropyrimidine 1 shows promise as a tool to provide spatiotemporal control over DNA structure on the miscrosecond timescale., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co(2+) over Pb(2+) compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co(2+) over Pb(2+) to levels observed in clone 11. A comparison of these sequences' secondary structures and Co(2+) selectivities suggested that metastable structures affect metal ion selectivity. The Co(2+) selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

8-17 is a DNAzyme with metal-dependent endoribonuclease activity. Recently, a variant termed 8-17NG was reported as the first nucleic acid enzyme capable of cleaving all 16 dinucleotide junctions of RNA with rate enhancements ranging from 1000- to 1,000,000,000-fold over background activity. We attributed this broad-ranging cleavage efficiency to global folding of the DNAzyme. We sought to examine the influence of dinucleotides at the cleavage site of 8-17NG on global folding by using three-color (3c) FRET. By comparing the folding of 8-17NG with all 16 possible dinucleotide junctions, we found all examined DNAzyme-substrate constructs adopted a two-step folding process in the presence of Mn(2+), which was consistent with previous metal-induced folding studies of 8-17. Interestingly, Mn(2+) titration experiments also suggest that the second folding step is dependent on dinucleotide identity: purine-purine junctions allowed 8-17NG to fold at lower concentrations than pyrimidine-pyrimidine linkages. This finding was corroborated by RNA cleavage assays, in which the largest improvement in cleavage yield was observed in pyrimidine-pyrimidine junctions when [Mn(2+)] was increased. Taken together, these results support the previously observed hierarchy of 8-17 activity for different cleavage sites. Complemented by earlier sequence and structure-function studies, this investigation allowed for the first detailed examination of crucial relationships between the structural influence and junction preferences of nucleic acid-catalyzed RNA cleavage reactions.

Enzymes play a crucial role in all living organisms by accelerating the rates of a myriad of biochemical reactions that are necessary to sustain life. Although the vast majority of known enzymes are made of protein, in recent years it has become increasingly apparent that other molecular formats, like nucleic acids, can also serve in this capacity. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid. These DNA-based enzymes offer the prospect of significant commercial utility, because they are exceptionally stable and can be produced very easily and inexpensively. The study of one particular DNAzyme, known as "8-17", has enhanced our understanding of DNAzyme-mediated catalysis. Moreover, the function of 8-17 has been regarded with special importance because it can catalyze sequence-specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields. In this review, we explore the creation, characterization, and application of the 8-17 RNA-cleaving DNAzyme.

Uranyl ion-specific DNAzyme: A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three-way-junction structure with catalytically important nucleotides residing in the bulge. A highly conserved GA mismatch is also crucial for the enzyme's activity.The biochemical characterization of a DNAzyme that is highly specific for uranyl (UO(2) (2+)) ions is described. Sequence alignment, enzyme truncation, and mutation studies have resulted in a conserved sequence that folds into a bulged stem-loop structure. Interestingly, a GA pair next to the scissile site is important for the uranyl ion-specific DNAzyme; this is reminiscent of the GT wobble base pair adjacent to the cleavage site that is crucial for the Pb(II)-specific 8-17 DNAzyme activity. Therefore wobble pairs might be important for formation of metal-specific metal-binding sites in DNAzymes. The DNAzyme binds the uranyl ion with a dissociation constant of 469 nM, which is among the strongest metal-binding affinities in nucleic acid enzymes reported to date. This explains why a catalytic beacon fluorescent sensor based on this enzyme has a detection limit (45 pM) that rivals the most-sensitive analytical instrument. It also has over 1 000 000-fold specificity for the uranyl ion over other metal ions. The DNAzyme can carry out multiple turnover reactions that follow the Michaelis-Menten equation, with a k(cat) of 1.46 min(-1) and a K(M) of 463 nM, similar to that of the 8-17 DNAzyme. The pH profile shows a bell-shaped curve that reaches a maximum at pH 5.5, at which the in vitro selection was carried out; this suggests that in vitro selection can be performed to obtain DNAzymes with optimal performance under specific conditions under which practical applications are required. These findings enrich our fundamental understanding of metal-binding sites in nucleic acids and allow the design of sensors with better performance.

Much interest has focused on the mechanisms of the five naturally occurring self-cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro-selected, RNA-cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self-cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell-shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro-selected, RNA-cleaving deoxyribozymes, such as the 8-17, 10-23, and 614.

We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.