ABSTRACT
Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising candidates for bioanalytical and biomedical applications due to their intriguing properties of enhanced biological stability and compatibility with rolling circle amplification. They are typically made from linear single-stranded (ss) DNA molecules via ligase-mediated ligation. However, it remains a great challenge to synthesize circular ssDNA molecules in high yield due to inherent side reactions where two or more of the same ssDNA molecules are ligated. Herein, we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a ligatable DNA aptamer that binds a DNA ligase and then by engineering this aptamer into a general-purpose templating DNA scaffold to guide the ligase to execute selective intramolecular circularization. We demonstrate the broad utility of this approach via the creation of several species of circular DNA molecules, including a circular DNAzyme sensor for a bacterium and a circular DNA aptamer sensor for a protein target with excellent detection sensitivity and specificity.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , DNA, Catalytic , DNA, Catalytic/metabolism , Aptamers, Nucleotide/chemistry , Ligases/metabolism , Nucleic Acid Amplification Techniques , DNA/chemistry , DNA, Circular , DNA, Single-StrandedABSTRACT
Real-time biomolecular monitoring requires biosensors based on affinity reagents, such as aptamers, with moderate to low affinities for the best binding dynamics and signal gain. We recently reported Pro-SELEX, an approach that utilizes parallelized SELEX and high-content bioinformatics for the selection of aptamers with predefined binding affinities. The Pro-SELEX pipeline relies on an algorithm, termed AptaZ, that can predict the binding affinities of selected aptamers. The original AptaZ algorithm is computationally complex and slows the overall throughput of Pro-SELEX. Here, we present Apta FastZ, a rapid equivalent of AptaZ. The Apta FastZ algorithm considers the spare nature of the sequences from SELEX and is coded to avoid unnecessary comparison between sequences. As a result, Apta FastZ achieved a 10 to 40-fold faster computing speed compared to the original AptaZ algorithm while maintaining identical outcomes, allowing the bioinformatics to be completed within 1-10 h for large-scale data sets. We further validated the affinity of myeloperoxidase aptamers predicted by Apta FastZ by experiments and observed a high level of linear correlation between predicted scores and measured affinities. Taken together, the implementation of Apta FastZ could greatly accelerate the current Pro-SELEX workflow, allowing customized aptamers to be discovered within 3 days using preselected DNA libraries.
Subject(s)
Aptamers, Nucleotide , Aptamers, Nucleotide/chemistry , SELEX Aptamer Technique , Gene Library , Computational BiologyABSTRACT
clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.
Subject(s)
Biosensing Techniques , Nucleic Acids , Humans , CRISPR-Cas Systems/genetics , RNA, Circular , DNA, Single-Stranded , RNAABSTRACT
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.â difficile but is too specific to recognize other pathogenic C.â difficile strains. To overcome this issue, herein we report RFD-CD2, another RFD that is not only highly specific to C.â difficile but also capable of recognizing diverse pathogenic C.â 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.â difficile at a concentration as low as 100â CFU/mL, thus making this DNAzyme an attractive molecular probe for rapid diagnosis of CDI caused by diverse strains of C.â difficile.
Subject(s)
Clostridioides difficile , Clostridium Infections , DNA, Catalytic , Humans , Clostridioides difficile/genetics , Clostridium Infections/diagnosis , Rapid Diagnostic TestsABSTRACT
Here we report on an ultra-sensitive colorimetric sensing platform that takes advantage of both the strong amplification power of rolling circle amplification (RCA) and the high efficiency of a simple urease-mediated litmus test. The presence of a target triggers the RCA reaction, and urease-labelled DNA can hybridize to the biotinylated RCA products and be immobilized onto streptavidin-coated magnetic beads. The urease-laden beads are then used to hydrolyze urea, leading to an increase in pH that can be detected by a simple litmus test. We show this sensing platform can be easily integrated with aptamers for sensing diverse targets via the detection of human thrombin and platelet-derived growth factor (PDGF) utilizing structure-switching aptamers as well as SARS-CoV-2 in human saliva using a spike-binding trimeric DNA aptamer. Furthermore, we demonstrate that this colorimetric sensing platform can be integrated into a simple paper-based device for sensing applications.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Humans , Urease , Colorimetry , DNA/metabolism , Nucleic Acid Amplification TechniquesABSTRACT
Engineering functional nucleic acids that are active under unusual conditions will not only reveal their hidden abilities but also lay the groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were "compelled" to accept 35 % dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35 % DMSO. This experiment led to the discovery of a new DNAzyme that requires 35 % DMSO for its catalytic activity and exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis, and its activity is enhanced by monovalent ions. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional, organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of catalytic DNA.
Subject(s)
DNA, Catalytic , Solvents , Dimethyl Sulfoxide , DNA, Catalytic/genetics , Ions , RNAABSTRACT
Reagent-free electronic biosensors capable of analyzing disease markers directly in unprocessed body fluids will enable the development of simple & affordable devices for personalized healthcare monitoring. Here we report a powerful and versatile nucleic acid-based reagent-free electronic sensing system. The signal transduction is based on the kinetics of an electrode-tethered molecular pendulum-a rigid double stranded DNA with one of the strands displaying an analyte-binding aptamer and the other featuring a redox probe-that exhibits field-induced transport modulated by receptor occupancy. Using chronoamperometry, which enables the sensor to circumvent the conventional Debye length limitation, the binding of an analyte can be monitored as these species increase the hydrodynamic drag. The sensing platform demonstrates a low femtomolar quantification limit and minimal cross-reactivity in analyzing cardiac biomarkers in whole blood collected from patients with chronic heart failure.
Subject(s)
Aptamers, Nucleotide , Nucleic Acids , Humans , Aptamers, Nucleotide/chemistry , DNA/chemistry , Electrodes , BiomarkersABSTRACT
Pathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori, and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.
Subject(s)
Aptamers, Nucleotide/metabolism , Bacteria/genetics , Biosensing Techniques/methods , DNA, Catalytic/metabolism , Aptamers, Nucleotide/chemistry , Bacteria/isolation & purification , DNA, Catalytic/chemistry , Escherichia coli/genetics , Escherichia coli/isolation & purification , Nucleic Acid Amplification Techniques , Point-of-Care SystemsABSTRACT
Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem-loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.
Subject(s)
Aptamers, Nucleotide/chemistry , DNA, Circular/chemistry , Thrombin/metabolism , Aptamers, Nucleotide/blood , Aptamers, Nucleotide/metabolism , DNA, Circular/blood , DNA, Circular/metabolism , G-Quadruplexes , Humans , Protein Binding , Thrombin/chemistryABSTRACT
Pen-side testing of farm animals for infectious diseases is critical for preventing transmission in herds and providing timely intervention. However, most existing pathogen tests have to be conducted in centralized labs with sample-to-result times of 2-4â days. Herein we introduce a test that uses a dual-electrode electrochemical chip (DEE-Chip) and a barcode-releasing electroactive aptamer for rapid on-farm detection of porcine epidemic diarrhea viruses (PEDv). The sensor exploits inter-electrode spacing reduction and active field mediated transport to accelerate barcode movement from electroactive aptamers to the detection electrode, thus expediting assay operation. The test yielded a clinically relevant limit-of-detection of 6â nM (0.37â µg mL-1 ) in saliva-spiked PEDv samples. Clinical evaluation of this biosensor with 12 porcine saliva samples demonstrated a diagnostic sensitivity of 83 % and specificity of 100 % with a concordance value of 92 % at an analysis time of one hour.
Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Coronavirus Infections/diagnosis , Coronavirus Infections/veterinary , DNA Barcoding, Taxonomic , Diarrhea/diagnosis , Diarrhea/veterinary , Porcine epidemic diarrhea virus/genetics , Saliva , Sensitivity and Specificity , Swine , Swine Diseases/diagnosisABSTRACT
Adenosine 5'-triphosphate (ATP) is a central extracellular signaling agent involved in various physiological and pathological processes. However, precise measurements of the temporal and spatial components of ATP dynamics are lacking due primarily to the limitations of available methods for ATP detection. Here, we report on the first effort to design a self-phosphorylating DNAzyme (SPDz) sensor for fluorescence imaging of ATP. In response to ATP, SPDz sensors exhibit subsecond response kinetics, extremely high specificity, and micromolar affinities. In particular, we demonstrate cell-surface-anchored SPDz sensors for fluorescence imaging of both stress-induced endogenous ATP release in astrocytes and mechanical stimulation-evoked ATP release at the single-cell level. We also validated their utility for visualizing the rapid dynamic properties of ATP signaling upon electrical stimulation in astrocytes. Thus, SPDz sensors are robust tools for monitoring ATP signaling underlying diverse cellular processes.
Subject(s)
Adenosine Triphosphate/chemistry , DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , Optical Imaging/methods , Single Molecule Imaging/methods , Astrocytes , Biosensing Techniques , Humans , MCF-7 Cells , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Phosphorylation , Sensitivity and Specificity , Stress, PhysiologicalABSTRACT
The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.
Subject(s)
Biosensing Techniques/methods , COVID-19 Testing/methods , COVID-19/virology , Electrochemical Techniques/methods , SARS-CoV-2/isolation & purification , Virion/isolation & purification , Biosensing Techniques/instrumentation , COVID-19 Testing/instrumentation , Electrochemical Techniques/instrumentation , Electrodes , Humans , Point-of-Care Testing , Saliva/virologyABSTRACT
There is a constant drive for affordable point-of-care testing (POCT) technologies for the detection of infectious human diseases. Herein, we report a simple platform for DNA detection that takes advantage of four techniques: commercially available pregnancy test strips (PTS), amplicon generation via loop-mediated isothermal amplification (LAMP), toehold-mediated strand displacement, and noncovalent immobilization of DNA on paper surface with DNA nanoflowers. This simple, separation-free platform is highly specific, as demonstrated with the detection of rtL180M, a single-nucleotide polymorphism observed in hepatitisâ B virus (HBV) associated with antiviral drug resistance. It is very sensitive, capable of detecting the targeted mutation at 2â copies µL-1 . It is able to correctly identify the unmutated and rtL180M genome types of HBV in clinical samples. Given its wide adaptability, we expect this platform can be easily modified for the detection of genetic variations associated with various pathogens and human diseases.
Subject(s)
DNA/analysis , Nanoparticles/chemistry , Female , Humans , Pregnancy , Pregnancy Tests , Sensitivity and SpecificityABSTRACT
Ribonucleaseâ I belongs to a class of nonspecific endoribonucleases and plays many important roles in a variety of biological and cellular processes. While their ubiquitous nature and high activity contribute to the well-known problem of RNase contamination in experimentation, their abundance in bacteria can potentially be leveraged as a biosensor target. As a result, there is substantial interest in generating a specific and reliable probe for RNase detection for a variety of purposes. To that end, we report on our unintentional discovery of the RNaseâ I probe RFA13-1 isolated through in vitro selection with the crude extracellular mixture from Clostridium difficile contaminated with Klebsiella aerogenes as a selection target. Characterization of RFA13-1 reveals that it exhibits high sensitivity to Escherichia coli RNaseâ I with a detection limit of 1.39â pm. Furthermore, RFA13-1 also shows high specificity for RNaseâ I produced only in select bacteria from the Enterobacteriaceae family. As a result, this probe offers a simple tool for RNaseâ I detection with potential applications in RNase functional studies, ribonuclease contamination monitoring, and bacterial detection.
Subject(s)
DNA Probes , Enterobacteriaceae/enzymology , Fluorescent Dyes , Ribonuclease, Pancreatic/isolation & purificationABSTRACT
The engineering of easy-to-use biosensors with ultra-low detection sensitivity remains a major challenge. Herein, we report a simple approach for creating such sensors through the use of an RNA-cleaving DNAzyme (RcD) and a strategy designed to concentrate its cleavage product significantly. The assay uses micron-sized beads loaded with a target-responsive RcD and a paper strip containing a microzone covered with a DNA oligonucleotide capable of capturing the cleavage product of the RcD through Watson-Crick hybridization. Placing the beads and the paper strip in a target-containing test sample allows the bead-bound RcD molecules to undergo target-induced RNA cleavage, releasing a DNA fragment that is captured by the paper strip. This strategy, though simple, is very effective in achieving high levels of detection sensitivity, being able to enrich the concentration of the cleavage product by three orders of magnitude. It is also compatible with both fluorescence-based and colorimetric reporting mechanisms. This work provides a simple platform for developing ultrasensitive biosensors that take advantage of the widely available RcDs as molecular recognition elements.
Subject(s)
Biosensing Techniques , DNA, Catalytic/chemistry , Nanotechnology , Oligodeoxyribonucleotides/chemistry , RNA/chemistry , Escherichia coli , Nucleic Acid Hybridization , RNA CleavageABSTRACT
Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxinâ B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxinâ B, fresh toxinâ B, and toxinâ B spiked into human stool samples. DNA aptamers selected using intact recombinant toxinâ B failed to recognize degraded toxinâ B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxinâ B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.
Subject(s)
Aptamers, Nucleotide/metabolism , Biosensing Techniques/methods , Peptide Fragments/metabolism , Proteolysis , Feces/chemistry , Humans , Paper , Toxins, Biological/analysis , Toxins, Biological/chemistry , Toxins, Biological/metabolismABSTRACT
Herein, we report on the design of a programmable DNA ribbon using long-chain DNA molecules with a user-defined repetitive padlock sequence. The DNA ribbon can be further combined with gold nanoparticles (AuNPs) to create a composite nanomaterial that contains an AuNP core and a high-density DNA crown carrying a cancer-cell-targeting DNA aptamer, a fluorescent tag for location tracking, and a cell-killing drug. This composite material can be efficiently internalized by cancer cells and its cellular location can be tracked by fluorescence imaging. The system offers several attractive characteristics, including simple design, tunable DNA crown, high drug-loading capacity, selective cell targeting, and pH-sensitive drug release. These features make such a material a promising therapeutic agent.
Subject(s)
Antineoplastic Agents/administration & dosage , DNA/chemistry , Drug Delivery Systems , Gold/chemistry , Metal Nanoparticles/chemistry , Neoplasms/drug therapy , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Doxorubicin/administration & dosage , Doxorubicin/therapeutic use , Humans , Microscopy, Atomic ForceABSTRACT
DNA-based enzymes, or DNAzymes, are not known to exist in Nature but can be isolated from random-sequence DNA pools using test tube selection techniques. Since the report of the first DNAzyme in 1994, many catalytic DNA molecules for catalyzing wide-ranging chemical transformations have been isolated and studied. Our laboratory has a keen interest in searching for diverse DNAzymes capable of cleaving RNA-containing substrates, determining their sequence requirements and structural properties, and examining their potential as biosensors. This Account begins with the description of an accidental discovery on the sequence adaptability of a small DNAzyme known as "8-17", when we performed 16 parallel selections to search for DNAzymes that targeted each and every possible dinucleotide junction of RNA for cleavage. DNAzyme 8-17 dominated all the selection pools targeting purine-containing junctions. In-depth sequence analysis revealed that 8-17 could manifest itself in many sequence options defined by the requirement of four absolutely conserved nucleotides. This study also exposed the fact that 8-17 had poor activity toward pyrimidine-pyrimidine junctions. With this information in hand, we proceeded to the discovery of diverse non-8-17 DNAzymes that exhibited robust catalytic activity under physiological conditions. These DNAzymes were found to universally interact with their substrates through two Watson-Crick binding arms and have a catalytic core of varying length and secondary-structure complexity. RNA-cleaving DNAzymes were also isolated to function at acidic conditions (pH 3-5), and these molecules exhibited intriguing pH profiles, with the highest activity precisely matching the pH used for their selection. Interestingly, these DNAzymes appear to use non-Watson-Crick interactions in defining their structures. More recently, we have embarked on the development of ligand-responsive RNA-cleaving fluorogenic DNAzymes that can recognize specific bacterial pathogens, such as Escherichia coli and Clostridium difficile, using a method that does not require a priori identification of a specific biomarker. Instead, the crude extracellular mixture as a whole is used as the target to drive the DNAzyme isolation. High recognition specificity can be achieved with a double-selection approach in which a DNA library is negatively selected against the cellular mixture prepared from unintended bacteria, followed by positive selection against the same mixture derived from a specific species or strain of bacterial pathogen. Finally, we have shown that DNAzymes' compatibility with DNA replication can benefit the design of amplification mechanisms that uniquely link the action of RNA-cleaving DNAzymes to rolling circle amplification, an isothermal DNA amplification technique. These methods are well suited for translating the target-binding and cleavage activity of an analyte-activated RNA-cleaving DNAzyme into the production of massive amounts of DNA amplicons to achieve ultrahigh detection sensitivity. Given the high chemical stability of DNA, our ability to discover catalytic DNA sequences by simultaneously evaluating as many as 1016 different DNA sequences, the accessibility to diverse RNA-cleaving DNAzymes in a single DNA pool, and the availability of methods for designing simple biosensors that incorporate RNA-cleaving DNAzymes, we believe we are moving closer to employing RNA-cleaving DNAzymes for exciting applications, such as point of care diagnostics or field detection of environmental toxins.
Subject(s)
Biosensing Techniques , DNA, Catalytic/metabolism , RNA/metabolism , DNA/chemistry , Nucleic Acid Amplification TechniquesABSTRACT
DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver inâ situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for inâ situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence inâ situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/biosynthesis , DNA/chemistry , MicroRNAs/analysis , Biocatalysis , DNA-Directed DNA Polymerase/chemistry , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , MCF-7 Cells , MicroRNAs/metabolism , Optical ImagingABSTRACT
DNAzymes refer to single-stranded DNA molecules with catalytic activity and can be isolated from synthetic random-sequence DNA pools using the technique of in vitro selection. DNAzymes that cleave RNA, known as "RNA-cleaving DNAzymes", represent one of the best-studied classes of DNAzymes and have been widely used for the development of biosensors and bioassays for various analytes. We have been interested in developing RNA-cleaving DNAzymes as bacterial sensors and these DNAzymes are engineered to perform three linked functions: recognition of a bacterial biomarker, RNA cleavage, and fluorescence generation. These fluorogenic DNAzymes emit fluorescence automatically in the presence of a bacterium of interest and can be used to set up a simple "mix-and-read" assay to detect this bacterium. In this article, we will discuss this DNAzyme system and present a proven strategy for isolating highly specific bacteria-responding DNAzyme probes from random-sequence DNA pools. We will also provide an in vitro selection protocol successfully used to derive RNA-cleaving fluorogenic DNAzyme probes that are capable of recognizing a targeted strain of Clostridium difficile.