In vitro evolution of ribonucleases from expanded genetic alphabets.

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid-base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

Self-Powered Engineering of Cell Membrane Receptors to On-Demand Regulate Cellular Behaviors.

On-demand engineering of cell membrane receptors to nongenetically intervene in cellular behaviors is still a challenge. Herein, a membraneless enzyme biofuel cell-based self-powered biosensor (EBFC-SPB) was developed for autonomously and precisely releasing Zn2+ to initiate DNAzyme-based reprogramming of cell membrane receptors, which further mediates signal transduction to regulate cellular behaviors. The critical component of EBFC-SPB is a hydrogel film on a biocathode which is prepared using a Fe3+-cross-linked alginate hydrogel film loaded with Zn2+ ions. In the working mode in the presence of glucose/O2, the hydrogel is decomposed due to the reduction of Fe3+ to Fe2+, accompanied by rapid release of Zn2+ to specifically activate a Zn2+-responsive DNAzyme nanodevice on the cell surface, leading to the dimerization of homologous or nonhomologous receptors to promote or inhibit cell proliferation and migration. This EBFC-SPB platform provides a powerful "sensing-actuating-treating" tool for chemically regulating cellular behaviors, which holds great promise in precision biomedicine.

Cleaving Folded RNA with DNAzyme Agents.

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 (Tm ) 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 Mg2+ at 37 °C. Unexpectedly, one optimized Dz had each RNA-binding arm with a Tm ≥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.

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.

DNA Nanoflower Eye Drops with Antibiotic-Resistant Gene Regulation Ability for MRSA Keratitis Target Treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm-associated bacterial keratitis is highly intractable, with strong resistance to ß-lactam antibiotics. Inhibiting the MRSA resistance gene mecR1 to downregulate penicillin-binding protein PBP2a has been implicated in the sensitization of ß-lactam antibiotics to MRSA. However, oligonucleotide gene regulators struggle to penetrate dense biofilms, let alone achieve efficient gene regulation inside bacteria cells. Herein, an eye-drop system capable of penetrating biofilms and targeting bacteria for chemo-gene therapy in MRSA-caused bacterial keratitis is developed. This system employed rolling circle amplification to prepare DNA nanoflowers (DNFs) encoding MRSA-specific aptamers and mecR1 deoxyribozymes (DNAzymes). Subsequently, ß-lactam antibiotic ampicillin (Amp) and zinc oxide (ZnO) nanoparticles are sequentially loaded into the DNFs (ZnO/Amp@DNFs). Upon application, ZnO on the surface of the nanosystem disrupts the dense structure of biofilm and fully exposes free bacteria. Later, bearing encoded aptamer, the nanoflower system is intensively endocytosed by bacteria, and releases DNAzyme under acidic conditions to cleave the mecR1 gene for PBP2a down-regulation, and ampicillin for efficient MRSA elimination. In vivo tests showed that the system effectively cleared bacterial and biofilm in the cornea, suppressed proinflammatory cytokines interleukin 1ß ï¼ˆIL-1ß） and tumor neocrosis factor-alpha （TNF-α）, and is safe for corneal epithelial cells. Overall, this design offers a promising approach for treating MRSA-induced keratitis.

In Vitro Selection and Characterization of a DNAzyme Probe for Diverse Pathogenic Strains of Clostridium difficile.

Clostridium difficile frequently causes an infectious disease known as Clostridium difficile infection (CDI), and there is an urgent need for the development of more effective rapid diagnostic tests for CDI. Previously we have developed an RNA-cleaving fluorogenic DNAzyme (RFD) probe, named RFD-CD1, that is capable of detecting a specific strain of C. difficile but is too specific to recognize other pathogenic C. difficile strains. To overcome this issue, herein we report RFD-CD2, another RFD that is not only highly specific to C. difficile but also capable of recognizing diverse pathogenic C. difficile strains. Extensive sequence and structure characterization establishes a pseudoknot structure and a significantly minimized sequence for RFD-CD2. As a fluorescent sensor, RFD-CD2 can detect C. difficile at a concentration as low as 100 CFU/mL, thus making this DNAzyme an attractive molecular probe for rapid diagnosis of CDI caused by diverse strains of C. difficile.

Unraveling the Kinetics of the 10-23 RNA-Cleaving DNAzyme.

DNA-based enzymes, or DNAzymes, are single-stranded DNA sequences with the ability to catalyze various chemical reactions, including the cleavage of the bond between two RNA nucleotides. Lately, an increasing interest has been observed in these RNA-cleaving DNAzymes in the biosensing and therapeutic fields for signal generation and the modulation of gene expression, respectively. Additionally, multiple efforts have been made to study the effects of the reaction environment and the sequence of the catalytic core on the conversion of the substrate into product. However, most of these studies have only reported alterations of the general reaction course, but only a few have focused on how each individual reaction step is affected. In this work, we present for the first time a mathematical model that describes and predicts the reaction of the 10-23 RNA-cleaving DNAzyme. Furthermore, the model has been employed to study the effect of temperature, magnesium cations and shorter substrate-binding arms of the DNAzyme on the different kinetic rate constants, broadening the range of conditions in which the model can be exploited. In conclusion, this work depicts the prospects of such mathematical models to study and anticipate the course of a reaction given a particular environment.

RNA-cleaving DNAzymes as a diagnostic and therapeutic agent against antimicrobial resistant bacteria.

The development of nucleic-acid-based antimicrobials such as RNA-cleaving DNAzyme (RCD), a short catalytically active nucleic acid, is a promising alternative to the current antibiotics. The current rapid spread of antimicrobial resistance (AMR) in bacteria renders some antibiotics useless against bacterial infection, thus creating the need for alternative antimicrobials such as DNAzymes. This review summarizes recent advances in the use of RCD as a diagnostic and therapeutic agent against AMR. Firstly, the recent diagnostic application of RCD for the detection of bacterial cells and the associated resistant gene(s) is discussed. The next section summarises the therapeutic application of RCD in AMR bacterial infections which includes direct targeting of the resistant genes and indirect targeting of AMR-associated genes. Finally, this review extends the discussion to challenges of utilizing RCD in real-life applications, and the potential of combining both diagnostic and therapeutic applications of RCD into a single agent as a theranostic agent.

Enzyme-Loaded Hemin/G-Quadruplex-Modified ZIF-90 Metal-Organic Framework Nanoparticles: Bioreactor Nanozymes for the Cascaded Oxidation of N-hydroxy-l-arginine and Sensing Applications.

Biocatalytic cascades are challenging to operate in homogeneous solution, where diffusional mass transport hinders efficient communication between the reactive components. There is great interest in developing devices to perform such transformations in confined environments, which increase the efficiency of the cascaded process by generating high local concentrations of the reactive species. Herein, a bioreactor-nanozyme assembly is introduced for the cascaded aerobic oxidation of N-hydroxy-l-arginine (NOHA) to citrulline in the presence of glucose. The reaction mimics a key step in the nitric oxide synthase oxidation of l-arginine in nature. The system consists of glucose oxidase (GOx)-loaded hemin/G-quadruplex (hemin/G4)-modified ZIF-90 metal-organic framework nanoparticles. The aerobic oxidation of glucose by GOx yields H2 O2 that fuels the hemin/G4-catalyzed oxidation of NOHA into citrulline. The process driven by the bioreactor-nanozyme system is ≈sixfold enhanced compared to the homogeneous mixture of the biocatalysts, due to its operation in the confined environment of the nanoparticles. Extension to a three-step cascade is then demonstrated using a bioreactor composed of ß-galactosidase/GOx-loaded hemin/G4-modified ZIF-90 nanoparticles activating the cascaded oxidation of NOHA to citrulline, in the presence of lactose. Moreover, the bioreactor-nanozyme hybrid is applied as a functional optical sensor of glucose, using fluorescence or chemiluminescence as readout signals.

A Versatile Electrochemical Biosensor for the Detection of Circulating MicroRNA toward Non-Small Cell Lung Cancer Diagnosis.

Circulating microRNAs (miRNAs) can be used as noninvasive biomarkers and are also found circulating in body fluids such as blood. Dysregulated miRNA expression is associated with many diseases, including non-small cell lung cancer (NSCLC), and the miRNA assay is helpful in cancer diagnosis, prognosis, and monitoring. In this work, a versatile electrochemical biosensing system is developed for miRNA detection by DNAzyme-cleavage cycling amplification and hybridization chain reaction (HCR) amplification. With cleavage by Mn2+ targeted DNAzyme, DNA-walker can move along the predesigned DNA tracks and contribute to the transduction and enhancement of signals. For the electrochemical process, the formation of multiple G-quadruplex-incorporated long double-stranded DNA (dsDNA/G-quadruplex) structures is triggered through HCR amplification. The introduction of G-quadruplex allows sensitive measurement of miRNA down to 5.68 fM with good specificity. Furthermore, by profiling miRNA in the NSCLC cohort, this designed strategy shows high efficiency (area under the curve (AUC) of 0.879 using receiver operating characteristic (ROC) analysis) with the sensitivity of 80.0% for NSCLC early diagnosis (stage I). For the discrimination of NSCLC and benign disease, the assay displays an AUC of 0.907, superior to six clinically-acceptable protein tumor markers. Therefore, this platform holds promise in clinical application toward NSCLC diagnosis and prognosis.

Core-Shell DNA-Cholesterol Nanoparticles Exert Lysosomolytic Activity in African Trypanosomes.

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.

Regulating Spatial Localization and Reactivity Biasness of DNAzymes by Metal Ions and Oligonucleotides.

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 (Hg2+ ) inhibitor is low. Interestingly, this biased localization can be broken by introduction of Hg2+ 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.

Selection of M2+ -Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine.

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+ ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dUga TP) and 5-aminoallyl-2'-dCTP (dCaa TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

DNA-assisted site-selective protein modification.

Protein modification is important for various types of biomedical research, including proteomics and therapeutics. Many methodologies for protein modification exist, but not all possess the required level of efficiency and site selectivity. This review focuses on the use of DNA to achieve the desired conversions and levels of accuracy in protein modification by using DNA (i) as a template to help concentrate dilute reactants, (ii) as a guidance system to achieve selectivity by binding specific proteins, and (iii) even as catalytic entity or construct to enhance protein modification reactions.

Inorganic nanoparticles coupled to nucleic acid enzymes as analytical signal amplification tools.

Nucleic acid enzymes (NAzymes) are a class of nucleic acid molecules with catalytic activity, which can be modulated by the presence of different species such as metal ions, genetic biomarkers, small molecules or proteins, among others. NAzymes offer several important advantages for development of novel bioanalytical strategies, resulting from their functionality as specific recognition elements and as amplified analytical signal generators, making them ideal candidates for developing highly specific bioanalytical strategies for the detection of a wide variety of targets. When coupled with the exceptional features of inorganic nanoparticles (NPs), the sensitivity of the assays can be significantly improved, allowing the detection of targets using many different detection techniques including visual readout, spectrophotometry, fluorimetry, electrochemiluminescence, voltammetry, and single-particle inductively coupled plasma-mass spectrometry. Here we provide an overview of the fundamentals of novel strategies developed to achieve analytical signal amplification based on the use of NAzymes coupled with inorganic NPs. Some representative examples of such strategies for the highly sensitive detection of different targets will be presented, including metal ions, proteins, DNA- or RNA-based biomarkers, and small molecules or microorganisms. Furthermore, future prospective challenges will be discussed.

An electrochemical aptasensor based on target triggered multiple-channel DNAzymes cycling amplification strategy with PtFe@Co-MOF as signal amplifier.

A novel electrochemical aptasensor for the detection of Aflatoxin B1 (AFB1) was developed for the first time by using the target-triggered multiple-channel deoxyribozymes (DNAzymes) cycling amplified assay with Pt Fe doped NH2-Co-MOF (PtFe@Co-MOF) as a signal amplifier. In the presence of AFB1, a self-assembling cross-over nucleic structure could be triggered by AFB1 via two aptamers' structure switching for strand displacement, resulting in four channels of Mg2+-dependent DNAzyme recycling simultaneously to multiply the detection signals. These DNAzymes cyclically split the substrate sequence to release the PtFe@Co-MOF labeled detection probe (DP), which is subsequently hybridized with the capture probes on the Au-deposited glassy carbon electrode. The fabrication procedure was characterized by differential pulse voltammetry, and the results of the morphological and element composition characteristics methods were analyzed to determine the successful preparation of PtFe@Co-MOF. The limit of detection (LOD) for AFB1 detection was 2 pg mL-1 with a linear range from 5 pg mL-1 to 80 ng mL-1. By comparison, the enhanced detection sensitivity has been found to originate from the efficient shearing of DNAzymes, enhanced peroxidase-like capability, and multiple active sites of PtFe@Co-MOF. Besides, this aptasensor showed high specificity for AFB1 compared with similar mycotoxins and exhibited high accuracy with low experimental cost and easy operation. Furthermore, the unique design of electrochemical aptasensors could provide a promising platform for the onsite determination of AFB1, as well as other targets by replacing the aptamer and other core recognition sequences.

A Practical Electrochemical Nanotool for Facile Quantification of Amino Acids in Single Cell.

Though significant advances are made in the arena of single-cell electroanalysis, quantification of intracellular amino acids of human cells remains unsolved. Exemplified by l-histidine (l-His), this issue is addressed by a practical electrochemical nanotool synergizing the highly accessible nanopipette with commercially available synthetic DNAzyme. The fabricated nanotools are screened before operation of a single-use manner, and the l-His-provoked cleavage of the DNA molecules can be sensibly transduced by the ionic current rectification response, the intrinsic property of nanopipette governed by its interior surface charges. Regional distribution of cytosolic l-His level in human cells is electrochemically quantified for the first time, and time-dependent drug treatment effects are further revealed. This work unveils the possibility of electrochemistry for quantification of cytosolic amino acids of a spatial- and time-based manner and ultimately enables a better understanding of amino acid-involved events in living cells.

DNAzyme-Functionalized Nanomaterials: Recent Preparation, Current Applications, and Future Challenges.

DNAzyme-nanomaterial bioconjugates are a popular hybrid and have received major attention for diverse biomedical applications, such as bioimaging, biosensor development, cancer therapy, and drug delivery. Therefore, significant efforts are made to develop different strategies for the preparation of inorganic and organic nanoparticles (NPs) with specific morphologies and properties. DNAzymes functionalized with metal-organic frameworks (MOFs), gold nanoparticles (AuNPs), graphene oxide (GO), and molybdenum disulfide (MoS2 ) are introduced and summarized in detail in this review. Moreover, the focus is on representative examples of applications of DNAzyme-nanomaterials over recent years, especially in bioimaging, biosensing, phototherapy, and stimulation response delivery in living systems, with their several advantages and drawbacks. Finally, the perspective regarding the future directions of research addressing these challenges is also discussed and highlighted.

Photoactivated Self-Disassembly of Multifunctional DNA Nanoflower Enables Amplified Autophagy Suppression for Low-Dose Photodynamic Therapy.

Low-dose photodynamic therapy (PDT) holds great promise for reducing undesired patient photosensitivity in cancer treatment. Yet, its therapeutic effect is significantly affected by intracellular cytoprotective processes, such as autophagy. Here, an efficient autophagy suppressor is developed, which is a multifunctional DNA nanoflower (DNF) consisted of tumor-targeting aptamers and DNAzymes for silencing autophagy-related genes, with surface modification of low-dose photosensitizer (Ce6). It is found that the multifunctional DNF can specifically target tumor cells and generate reactive oxygen species (ROS) under light irradiation to trigger self-disassembly of DNF, enhancing the bioavailability of encoded DNAzymes, leading to amplified autophagy suppression. As a facile spatiotemporally programmable photogene therapy platform, the designed DNF is able to suppress tumor growth in vivo with a very low injection dose of Ce6 (18 µg kg-1 , around 100 times lower than the generally applied dose), representing a promising strategy for cancer therapy with safely low-dose PDT.

Functional Nucleic Acids Under Unusual Conditions.

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.