Detection of SARS-CoV-2 using machine learning-enabled paper-assisted ratiometric fluorescent sensors based on target-induced magnetic DNAzyme.

The development of an advanced analytical platform with regard to SARS-CoV-2 is crucial for public health. Herein, we present a machine learning platform based on paper-assisted ratiometric fluorescent sensors for highly sensitive detection of the SARS-CoV-2 RdRp gene. The assay involves target-induced rolling circle amplification to generate magnetic DNAzyme, which is then detectable using the paper-assisted ratiometric fluorescent sensor. This sensor detects the SARS-CoV-2 RdRp gene with a visible-fluorescence color response. Moreover, leveraging different fluorescence responses, the ResNet algorithm of machine learning assists in accurately identifying fluorescence images and differentiating the concentration of the SARS-CoV-2 RdRp gene with over 99% recognition accuracy. The machine learning platform exhibits exceptional sensitivity and color responsiveness, achieving a limit of detection of 30 fM for the SARS-CoV-2 RdRp gene. The integration of intelligent artificial vision with the paper-assisted ratiometric fluorescent sensor presents a novel approach for the on-site detection of COVID-19 and holds potential for broader use in disease diagnostics in the future.

Multicomponent DNAzyme Nanomachines: Structure, Applications, and Prospects.

Nucleic acids (NAs) are important components of living organisms responsible for the storage and transmission of hereditary information. They form complex structures that can self-assemble and bind to various biological molecules. DNAzymes are NAs capable of performing simple chemical reactions, which makes them potentially useful elements for creating DNA nanomachines with required functions. This review focuses on multicomponent DNA-based nanomachines, in particular on DNAzymes as their main functional elements, as well as on the structure of DNAzyme nanomachines and their application in the diagnostics and treatment of diseases. The article also discusses the advantages and disadvantages of DNAzyme-based nanomachines and prospects for their future applications. The review provides information about new technologies and the possibilities of using NAs in medicine.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

Dual biomarkers-activatable hollow MnO2-Based theranostic nanoplatform for efficient breast cancer-specific multisite fluorescence imaging and synergistic therapy.

BACKGROUND: Theranostic nanoplatforms with integrated diagnostic imaging and multiple therapeutic functions play a vital role in precise diagnosis and efficient treatment for breast cancer, but unfortunately, these nanoplatforms are usually stuck in single-site imaging and single mode of treatment, causing unsatisfactory diagnostic and therapeutic efficiency. Herein, a dual biomarkers-activatable facile hollow mesoporous MnO2 (H-MnO2)-based theranostic nanoplatform, DNAzyme@H-MnO2-MUC1 aptamer (DHMM), was constructed for the simultaneous multi-site diagnosis and multiple treatment of breast cancer. RESULTS: The DHMM acted as an integrated diagnostic and therapeutic nanoplatform that realizes multi-site fluorescence imaging-guided high-efficient photothermal/chemodynamic/gene synergistic therapy (PTT/CDT/GT) for breast cancer. The H-MnO2 exhibits high loading capacity for Cy5-MUC1 aptamer (3.05 pmoL µg-1) and FAM-DNAzyme (3.37 pmoL µg-1), and excellent quenching for the probes. In the presence of MUC1 on the cell membrane and GSH in the cytoplasm, Cy5-MUC1 aptamer and FAM-DNAzyme was activated triggering dual-channel fluorescence imaging at different sites. Moreover, the self-supplied Mn2+ was further supplied as DNAzyme cofactors to catalytic cleavage intracellular EGR-1 mRNA for high-efficient GT and stimulated the Fenton-like reaction for CDT. The H-MnO2 also showcases a favorable photothermal performance with a photothermal conversion efficiency of 44.16%, which ultimately contributes to multi-site fluorescence imaging-guided synergistic treatment with an apoptosis rate of 71.82%. SIGNIFICANCE: This dual biomarker-activatable multiple therapeutic nanoplatform was realized multi-site fluorescence imaging-guided PTT/CDT/GT combination therapy for breast cancer with higher specificity and efficiency, which provides a promising theranostic nanoplatform for the precision and efficiency of breast cancer treatment.

An enzyme-free sensing platform for miRNA detection and in situ imaging in clinical samples based on DNAzyme cleavage-triggered catalytic hairpin assembly.

MicroRNA (miRNA) is demonstrated to be associated with the occurrence and development of various diseases including cancer. Currently, most miRNA detection methods are confined to in vitro detection and cannot obtain information on the temporal and spatial expression of miRNA in relevant tissues and cells. In this work, we established a novel enzyme-free method that can be applied to both in vitro detection and in situ imaging of miRNA by integrating DNAzyme and catalytic hairpin assembly (CHA) circuits. This developed CHA-Amplified DNAzyme miRNA (CHAzymi) detection system can realize the quantitively in vitro detection of miR-146b (the biomarker of papillary thyroid carcinoma, PTC) ranging from 25 fmol to 625 fmol. This strategy has also been successfully applied to in situ imaging of miR-146b both in human PTC cell TPC-1 and clinical samples, showing its capacity as an alternative diagnostic method for PTC. Furthermore, this CHAzymi system can be employed as a versatile sensing platform for various miRNAs by revising the relevant sequences. The results imply that this system may expand the modality of miRNA detection and show promise as a novel diagnostic tool in clinical settings, providing valuable insights for effective treatment and management of the disease.

Label-free and portable detection of HIV-DNA by a handheld luminometer.

BACKGROUND: The human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers. RESULTS: In this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H2O2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05-10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA. SIGNIFICANCE: In-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings.

Isothermal Reciprocal Catalytic DNA Circuit for Sensitive Analysis of Kanamycin.

Inappropriate use of veterinary drugs can result in the presence of antibiotic residues in animal-derived foods, which is a threat to human health. A simple yet efficient antibiotic-sensing method is highly desirable. Programmable DNA amplification circuits have supplemented robust toolkits for food contaminants monitoring. However, they currently face limitations in terms of their intricate design and low signal gain. Herein, we have engineered a robust reciprocal catalytic DNA (RCD) circuit for highly efficient bioanalysis. The trigger initiates the cascade hybridization reaction (CHR) to yield plenty of repeated initiators for activating the rolling circle amplification (RCA) circuit. Then the RCA-generated numerous reconstituted triggers can reversely stimulate the CHR circuit. This results in a self-sufficient supply of numerous initiators and triggers for the successive cross-invasion of CHR and RCA amplifiers, thus leading to exponential signal amplification for the highly efficient detection of analytes. With its flexible programmability and modular features, the RCD amplifier can serve as a universal toolbox for the high-performance and accurate sensing of kanamycin in buffer and food samples including milk, honey, and fish, highlighting its enormous promise for low-abundance contaminant analysis in foodstuffs.

DNA Nanospheres Assisted Spatial Confinement Signal Amplification for MicroRNA Imaging in Live Cancer Cells.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

A triggered DNA nanomachine with enzyme-free for the rapid detection of telomerase activity in a one-step method.

BACKGROUND: Telomerase is considered a biomarker for the early diagnosis and clinical treatment of cancer. The rapid and sensitive detection of telomerase activity is crucial to biological research, clinical diagnosis, and drug development. However, the main obstacles facing the current telomerase activity assay are the cumbersome and time-consuming procedure, the easy degradation of the telomerase RNA template and the need for additional proteases. Therefore, it is necessary to construct a new method for the detection of telomerase activity with easy steps, efficient reaction and strong anti-interference ability. RESULTS: Herein, an efficient, enzyme-free, economical, sensitive, fluorometric detection method for telomerase activity in one-step, named triggered-DNA (T-DNA) nanomachine, was created based on target-triggered DNAzyme-cleavage activity and catalytic molecular beacon (CMB). Telomerase served as a switch and extended few numbers of (TTAGGG)n repeat sequences to initiate the signal amplification in the T-DNA nanomachine, resulting in a strong fluorescent signal. The reaction was a one-step method with a shortened time of 1 h and a constant temperature of 37 °C, without the addition of any protease. It also sensitively distinguished telomerase activity in various cell lines. The T-DNA nanomachine offered a detection limit of 12 HeLa cells µL-1, 9 SK-Hep-1 cells µL-1 and 3 HuH-7 cells µL-1 with a linear correlation detection range of 0.39 × 102-6.25 × 102 HeLa cells µL-1 for telomerase activity. SIGNIFICANCE: In conclusion, our study demonstrated that the triggered-DNA nanomachine fulfills the requirements for rapid detection of telomerase activity in one-step under isothermal and enzyme-free conditions with excellent specificity, and its simple and stable structure makes it ideal for complex systems. These findings indicated the application prospect of DNA nanomachines in clinical diagnostics and provided new insights into the field of DNA nanomachine-based bioanalysis.

Rectifying artificial nanochannels with multiple interconvertible permeability states.

Transmembrane channels play a vital role in regulating the permeation process, and have inspired recent development of biomimetic channels. Herein, we report a class of artificial biomimetic nanochannels based on DNAzyme-functionalized glass nanopipettes to realize delicate control of channel permeability, whereby the surface wettability and charge can be tuned by metal ions and DNAzyme-substrates, allowing reversible conversion between different permeability states. We demonstrate that the nanochannels can be reversibly switched between four different permeability states showing distinct permeability to various functional molecules. By embedding the artificial nanochannels into the plasma membrane of single living cells, we achieve selective transport of dye molecules across the cell membrane. Finally, we report on the advanced functions including gene silencing of miR-21 in single cancer cells and selective transport of Ca2+ into single PC-12 cells. In this work, we provide a versatile tool for the design of rectifying artificial nanochannels with on-demand functions.

Catalytic hairpin self-assembly amplification fluorescence detection of chloramphenicol based on cross-shaped DNA and UiO-66.

A catalytic hairpin self-assembly (CHA) amplification method was developed for CAP detection based on cross-shaped DNA and UiO-66. MOF was used to quench the fluorescent signal of FAM labeled DNA. Cross-shaped DNA with four fluorophore group (FAM) was utilized to enhance the fluorescent intensity. CAP could open hairpin structure of H-apt and induce CHA reaction. The product of CHA hybridized with cross-shaped DNA, resulting its leaving from the surface of UiO-66 and recovery of fluorescent signal. The limit of detection (LOD) was low to 0.87 pM. This method had been successfully applied for the detection of CAP in actual samples. Importantly, the high sensitivity was attributed to the great amplification efficiency of CHA, strong fluorescent intensity of cross-shaped DNA structure and great fluorescent quenched efficiency of UiO-66.

A dual-core 3D DNA nanomachine based on DNAzyme positive feedback loop for highly sensitive MicroRNA imaging in living cells.

A double 3D DNA walker nanomachine by DNAzyme self-driven positive feedback loop amplification for the detection of miRNA was constructed. This method uses two gold nanoparticles as the reaction core, and because of the spatial confinement effect the local concentration of the reactants increase the collision efficiency was greatly improved. Meanwhile, the introduction of positive feedback loop promotes the conversion efficiency. In presence of miRNA-21, a large amount of DNAzyme was released and hydrolyze the reporter probe, resulting the recovery of fluorescence signal. The linear range for miRNA-21 is 0.5-60 pmol/L, and the detection limit is 0.41 pmol/L (S/N = 3). This nanomachine has been successfully used for accurate detection of miRNA-21 expression levels in cell lysates. At the same time, it can enter cells for intracellular miRNA-21 fluorescence imaging, distinguishing tumor cells from normal cells. This combination of in vitro detection and imaging analysis of living cells can achieve the goal of jointly detecting cancer markers through multiple pathways, providing new ideas for early diagnosis and screening of diseases.

Multiply Guaranteed Catalytic DNA Circuit for Cancer-Cell-Selective Imaging of miRNA and Robust Evaluation of Drug Resistance.

Catalytic DNA circuits are desirable for sensitive bioimaging in living cells; yet, it remains a challenge to monitor these intricate signal communications because of the uncontrolled circuitry leakage and insufficient cell selectivity. Herein, a simple yet powerful DNA-repairing enzyme (APE1) activation strategy is introduced to achieve the site-specific exposure of a catalytic DNA circuit for realizing the selectively amplified imaging of intracellular microRNA and robust evaluation of the APE1-involved drug resistance. Specifically, the circuitry reactants are firmly blocked by the enzyme recognition/cleavage site to prevent undesirable off-site circuitry leakage. The caged DNA circuit has no target-sensing activity until its circuitry components are activated via the enzyme-mediated structural reconstitution and finally transduces the amplified fluorescence signal within the miRNA stimulation. The designed DNA circuit demonstrates an enhanced signal-to-background ratio of miRNA assay as compared with the conventional DNA circuit and enables the cancer-cell-selective imaging of miRNA. In addition, it shows robust sensing performance in visualizing the APE1-mediated chemoresistance in living cells, which is anticipated to achieve in-depth clinical diagnosis and chemotherapy research.

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100 pM and achieves a detection limit of 5 fM (3σ criterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

Self-Feedback DNAzyme Motor for Cascade-Amplified Imaging of mRNA in Live Cells and In Vivo.

DNA motors have attracted extensive interest in biosensing and bioimaging. However, the amplification capacity of the existing DNA motor systems is limited since the products from the walking process are unable to feedback into the original DNA motor systems. As a result, the sensitivities of such systems are limited in the contexts of biosensing and bioimaging. In this study, we report a novel self-feedback DNAzyme motor for the sensitive imaging of tumor-related mRNA in live cells and in vivo with cascade signal amplification capacity. Gold nanoparticles (AuNPs) are modified with hairpin-locked DNAzyme walker and track strands formed by hybridizing Cy5-labeled DNA trigger-incorporated substrate strands with assistant strands. Hybridization of the target mRNA with the hairpin strands activates DNAzyme and promotes the autonomous walking of DNAzyme on AuNPs through DNAzyme-catalyzed substrate cleavage, resulting in the release of many Cy5-labeled substrate segments containing DNA triggers and the generation of an amplified fluorescence signal. Moreover, each released DNA trigger can also bind with the hairpin strand to activate and operate the original motor system, which induces further signal amplification via a feedback mechanism. This motor exhibits a 102-fold improvement in detection sensitivity over conventional DNAzyme motors and high selectivity for target mRNA. It has been successfully applied to distinguish cancer cells from normal cells and diagnose tumors in vivo based on mRNA imaging. The proposed DNAzyme motor provides a promising paradigm for the amplified detection and sensitive imaging of low-abundance biomolecules in vivo.

Renewable Electrochemiluminescence Biosensor Based on Eu-MOGs as a Highly Efficient Emitter and a DNAzyme-Mediated Dual-drive DNA Walker as a Signal Amplifier for Ultrasensitive Detection of miRNA-222.

Herein, novel europium metal-organic gels (Eu-MOGs) with excellent cathode electrochemiluminescence (ECL) emission are first used to construct biosensors for the ultrasensitive detection of miRNA-222. Impressively, N and O elements of organic ligand 2,2':6,2″-terpyridine 4,4',4″-tricarboxylic acid (H3-tctpy) can perfectly coordinate with Eu3+ to form Eu-MOGs, which not only reduce nonradiative transition caused by the intramolecular free rotation of phenyl rings in other MOGs to enhance the ECL signal with extraordinary ECL efficiency as high as 37.2% (vs the [Ru(bpy)3]2+/S2O82- ECL system) but also reinforce ligand-to-metal charge transfer (LMCT) by the strong affinity between Eu3+ and N and O elements to greatly improve the stability of ECL signals. Besides, an improved nucleic acid cascade amplification reaction is developed to greatly raise the conversion efficiency from target miRNA-222 to a DNAzyme-mediated dual-drive DNA walker as output DNA, which can simultaneously shear the specific recognition sites from two directions. In that way, the proposed biosensor can further enhance the detection sensitivity of miRNA-222 with a linear range of 10 aM-1 nM and a detection limit (LOD) of 8.5 aM, which can also achieve an accurate response in cancer cell lysates of MHCC-97L and HeLa. Additionally, the biosensor can be self-regenerated by the folding/unfolding of related triplets with pH changes to simplify experimental operations and reduce the cost. Hence, this work proposed novel MOGs with stable and intense ECL signals for the construction of a renewable ECL biosensor, supplying a reliable detection method in biomarker analysis and disease diagnosis.

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/µL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

Modular Weaving DNAzyme in Skeleton of DNA Nanocages for Photoactivatable Catalytic Activity Regulation.

DNAzymes exhibit tremendous application potentials in the field of biosensing and gene regulation due to its unique catalytic function. However, spatiotemporally controlled regulation of DNAzyme activity remains a daunting challenge, which may cause nonspecific signal leakage or gene silencing of the catalytic systems. Here, we report a photochemical approach via modular weaving active DNAzyme into the skeleton of tetrahedral DNA nanocages (TDN) for light-triggered on-demand liberation of DNAzyme and thus conditional control of gene regulation activity. We demonstrate that the direct encoding of DNAzyme in TDN could improve the biostability of DNAzyme and ensure the delivery efficiency, comparing with the conventional surface anchoring strategy. Furthermore, the molecular weaving of the DNA nanostructures allows remote control of DNAzyme-mediated gene regulation with high spatiotemporal precision of light. In addition, we demonstrate that the approach is applicable for controlled regulation of the gene editing functions of other functional nucleic acids.

Chemoenzymatic Installation of Site-Specific Chemical Groups on DNA Enhances the Catalytic Activity.

Functional DNAs are valuable molecular tools in chemical biology and analytical chemistry but suffer from low activities due to their limited chemical functionalities. Here, we present a chemoenzymatic method for site-specific installation of diverse functional groups on DNA, and showcase the application of this method to enhance the catalytic activity of a DNA catalyst. Through chemoenzymatic introduction of distinct chemical groups, such as hydroxyl, carboxyl, and benzyl, at specific positions, we achieve significant enhancements in the catalytic activity of the RNA-cleaving deoxyribozyme 10-23. A single carboxyl modification results in a 100-fold increase, while dual modifications (carboxyl and benzyl) yield an approximately 700-fold increase in activity when an RNA cleavage reaction is catalyzed on a DNA-RNA chimeric substrate. The resulting dually modified DNA catalyst, CaBn, exhibits a kobs of 3.76 min-1 in the presence of 1 mM Mg2+ and can be employed for fluorescent imaging of intracellular magnesium ions. Molecular dynamics simulations reveal the superior capability of CaBn to recruit magnesium ions to metal-ion-binding site 2 and adopt a catalytically competent conformation. Our work provides a broadly accessible strategy for DNA functionalization with diverse chemical modifications, and CaBn offers a highly active DNA catalyst with immense potential in chemistry and biotechnology.

The Effect of 8,5'-Cyclo 2'-deoxyadenosine on the Activity of 10-23 DNAzyme: Experimental and Theoretical Study.

The in vivo effectiveness of DNAzymes 10-23 (Dz10-23) is limited due to the low concentration of divalent cations. Modifications of the catalytic loop are being sought to increase the activity of Dz10-23 in physiological conditions. We investigated the effect of 5'S or 5'R 5',8-cyclo-2'deoxyadenosine (cdA) on the activity of Dz10-23. The activity of Dz10-23 was measured in a cleavage assay using radiolabeled RNA. The Density Functional Tight Binding methodology with the self-consistent redistribution of Mulliken charge modification was used to explain different activities of DNAzymes. The substitution of 2'-deoxyadenosine with cdA in the catalytic loop decreased the activity of DNAzymes. Inhibition was dependent on the position of cdA and its absolute configuration. The order of activity of DNAzymes was as follows: wt-Dz > ScdA5-Dz ≈ RcdA15-Dz ≈ ScdA15-Dz > RcdA5-Dz. Theoretical studies revealed that the distance between phosphate groups at position 5 in RcdA5-Dz was significantly increased compared to wt-Dz, while the distance between O4 of dT4 and nonbonding oxygen of PO2 attached to 3'O of dG2 was much shorter. The strong inhibitory effect of RcdA5 may result from hampering the flexibility of the catalytic loop (increased rigidity), which is required for the proper positioning of Me2+ and optimal activity.