Nucleic Acid Enzymes Research Articles

Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging.

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

Solid-Phase PCR-Amplified DNAzyme Activity for Real-Time FO-SPR Detection of the MCR-2 Gene.

The polymerase chain reaction (PCR) has been the gold standard molecular analysis technique for decades and has seen quite some evolution in terms of reaction components, methodology, and readout mechanisms. Nucleic acid enzymes (NAzymes) have been used to further exploit the applications of PCR, but so far the work was limited to the colorimetric G-quadruplex or fluorescent substrate cleaving NAzymes. In this study, a solid-phase, fiber optic surface plasmon resonance (FO-SPR) technique is presented as an alternative readout for PCR utilizing NAzymes. First, the surface cleavage activity of DNAzyme-extended amplicons (DNAzyme-amps) is established, followed by optimization of the PCR conditions, which are required for compatibility with the FO-SPR system. Next, by integrating the complement of a 10-23 DNAzyme into the primer pair, PCR-amplified DNAzyme-amps were generated, tested, and validated on qPCR for the detection of the antimicrobial resistance gene MCR-2. Once validated, this primer concept was developed as a one-step assay, driven by PCR-amplified DNAzymes, for FO-SPR-based sensitive and specific detection. Using gold nanoparticle labeled RNA-DNA hybrid strands as substrate for the DNAzyme, PCR-amplified DNAzyme-amps generated in the presence of MCR-2 gene were monitored in real-time, which resulted in an experimental limit of detection of 4 × 105 copy numbers or 6.6 fM. In addition, the DNAzyme-based FO-PCR assay was able to discriminate between the MCR-1 and MCR-2 genes, to further prove the specificity of this assay. Henceforth, this DNAzyme-based fiber optic PCR assay provides a universally applicable, real-time system for the detection of virtually any target NA, in a specific and sensitive manner.

Functional nucleic acid-based fluorescence polarization/anisotropy biosensors for detection of biomarkers.

The sensitive and selective detection of biomarkers plays a crucial role in disease diagnostics, drug discovery, and early screening of cancers. The achievement of this goal highly depends on the continuous development of biosensing technologies. Among them, fluorescence anisotropy/polarization (FA/FP) analysis receives increasing interest due to the advantage of simple operation, fast response, and no background interference. In recent decades, great progress has been achieved in FA/FP sensors thanks to the development of functional nucleic acids (FNAs) including aptamers and nucleic acid enzymes. This review focuses on FNA-based FA/FP sensors for the quantitative detection of biomarkers, such as nucleic acid, small molecules, and proteins. The design strategies, recognition elements, and practical applications are fully highlighted. The article also discusses the challenges of applying FNA-based FA/FP sensors in the next generation and the potential solutions along with future prospects. Graphical abstract.

One-step isothermal RNA detection with LNA-modified MNAzymes chaperoned by cationic copolymer

RNA detection permits early diagnosis of several infectious diseases and cancers, which prevent propagation of diseases and improve treatment efficacy. However, standard technique for RNA detection such as reverse transcription-quantitative polymerase chain reaction has complicated procedure and requires well-trained personnel and specialized lab equipment. These shortcomings limit the application for point-of-care analysis which is critical for rapid and effective disease management. The multicomponent nucleic acid enzymes (MNAzymes) are one of the promising biosensors for simple, isothermal and enzyme-free RNA detection. Herein, we demonstrate simple yet effective strategies that significantly enhance analytical performance of MNAzymes. The addition of the cationic copolymer and structural modification of MNAzyme significantly enhanced selectivity and activity of MNAzymes by 250 fold and 2,700 fold, respectively. The highly simplified RNA detection system achieved a detection limit of 73 fM target concentration without additional amplification. The robustness of MNAzyme in the presence of non-target RNA was also improved. Our finding opens up a route toward the development of an alternative rapid, sensitive, isothermal, and protein-free RNA diagnostic tool, which expected to be of great clinical significance.

Transcriptome Analysis Reveals Sexual Disparities in Differential Gene Expression Signature in Rat Brain Microvessels

BackgroundSex appears to be an important determinant of brain microvessel (MV) function and susceptibility to cerebrovascular and neurological diseases. However, little is known about sex differences in brain MVs. The object of the current study was to examine differential gene expression in brain MVs of young male and female rats.MethodsMicrovessels (MVs) were isolated from the brains of age‐matched, male and female, Sprague‐Dawley rats (8–10 weeks old, n = 3/group). The quality of the isolated MVs were monitored under light microscopy, and the presence of end‐arterioles, capillaries, and venules in MVs were confirmed by alkaline phosphatase staining. Total RNA was isolated, and RNASeq analysis was performed by Illumina NextSeq 550 sequencing system. Banana SLUG analytics platform from UCSC was used for bioinformatics analysis of the 23,786 identified genes.ResultsA total of 298 (1.2%) significant (FDR < 0.05) differentially expressed (DE) genes were identified, of which 40% (119/298) and 60% (179/298) of DE genes were abundant in male and female MVs respectively. GO analysis categorized under biological process, cellular compartment and molecular functions to discover the most important putative functions of the target genes. Using PANTHER protein class, 23 most significant (FDR < 0.05) gene functions were higher in male than female MVs. Nucleic acid binding, enzyme modulator, and transcription factor were the top three gene functions, abundantly high in male MVs, in which more than 20 significant DE genes were involved in each class. Based on Reactome and KEGG enrichment analysis, 20 significantly (FDR<0.05) enriched canonical pathways were abundantly high in male rat MVs. Synthesis of glycosylphosphatidylinositol (GPI), post‐translational modification of GPI‐anchored proteins, and GPI‐anchor biosynthesis were the top three significantly enriched canonical pathways that were abundantly high in male rat MVs. Similarly, peptide chain elongation, ribosome, and 3′‐UTR‐mediated translational regulation were the top three significantly (FDR<0.05) enriched canonical pathways that were abundantly high in female rat MVs. Genes involved in different biological processes like vasodilation, mannosylation, mitochondria morphogenesis, anchor metabolic process, t‐RNA transport, DNA replication, and basal transcription factors were predominantly high in male rat MVs. Surprisingly and simultaneously, genes involved in mitochondrial apoptotic pathway, necroptotic processes, FAS, and caspase activation pathways were also abundantly higher in male than female MVs.ConclusionsSeveral DE genes and signaling pathways were identified in the brain MVs of male and female rats. Our findings show major differences in gene expression of male and female rats and provide a foundation to study the role of brain MVs in the development of cerebrovascular and other neurological diseases.Support or Funding InformationHL‐093554

Splitting aptamers and nucleic acid enzymes for the development of advanced biosensors.

In analogy to split-protein systems, which rely on the appropriate fragmentation of protein domains, split aptamers made of two or more short nucleic acid strands have emerged as novel tools in biosensor set-ups. The concept relies on dissecting an aptamer into a series of two or more independent fragments, able to assemble in the presence of a specific target. The stability of the assembled structure can further be enhanced by functionalities that upon folding would lead to covalent end-joining of the fragments. To date, only a few aptamers have been split successfully, and application of split aptamers in biosensing approaches remains as promising as it is challenging. Further improving the stability of split aptamer target complexes and with that the sensitivity as well as efficient working modes are important tasks. Here we review functional nucleic acid assemblies that are derived from aptamers and ribozymes/DNAzymes. We focus on the thrombin, the adenosine/ATP and the cocaine split aptamers as the three most studied DNA split systems and on split DNAzyme assemblies. Furthermore, we extend the subject into split light up RNA aptamers used as mimics of the green fluorescent protein (GFP), and split ribozymes.

Tautomerism of Guanine Analogues.

Tautomerism of nucleic acid (NA) bases is a crucial factor for the maintenance and translation of genetic information in organisms. Only canonical tautomers of NA bases can form hydrogen-bonded complexes with their natural counterparts. On the other hand, rare tautomers of nucleobases have been proposed to be involved in processes catalysed by NA enzymes. Isocytosine, which can be considered as a structural fragment of guanine, is known to have two stable tautomers both in solution and solid states. The tautomer equilibrium of isocytosine contrasts with the remarkable stability of the canonical tautomer of guanine. This paper investigates the factors contributing to the stability of the canonical tautomer of guanine by a combination of NMR experiments and theoretical calculations. The electronic effects of substituents on the stability of the rare tautomers of isocytosine and guanine derivatives are studied by density functional theory (DFT) calculations. Selected derivatives are studied by variable-temperature NMR spectroscopy. Rare tautomers can be stabilised in solution by intermolecular hydrogen-bonding interactions with suitable partners. These intermolecular interactions give rise to characteristic signals in proton NMR spectra, which make it possible to undoubtedly confirm the presence of a rare tautomer.

DNA-only, microwell-based bioassay for multiplex nucleic acid detection with single base-pair resolution using MNAzymes

In disease diagnostics, single- and multiplex nucleic acid (NA) detection, with the potential to discriminate mutated strands, is of paramount importance. Current techniques that rely on target amplification or protein-enzyme based signal amplification are highly relevant, yet still plagued by diverse drawbacks including erroneous target amplification, and the limited stability of protein enzymes. As a solution, we present a multicomponent nucleic acid enzymes (MNAzymes)-based system for singleplex and multiplex detection of NA targets in microwells down to femtomolar (fM) concentrations, without the need for any target amplification or protein enzymes, while operating at room temperature and with single base-pair resolution. After successful validation of the MNAzymes in solution, their performance was further verified on beads in bulk and in femtoliter-sized microwells. The latter is not only a highly simplified system compared to previous microwell-based bioassays but, with the detection limit of 180 fM, it is to-date the most sensitive NAzyme-mediated, bead-based approach, that does not rely on target amplification or any additional signal amplification strategies. Furthermore, we demonstrated, for the first time, multiplexed target detection in microwells, both from buffer and nasopharyngeal swab samples, and presented superior single base-pair resolution of this assay. Because of the design flexibility of MNAzymes and direct demonstration in swab samples, this system holds great promise for multiplexed detection in other clinically relevant matrices without the need for any additional NA or protein components. Moreover, these findings open up the potential for the development of next-generation, protein-free diagnostic tools, including digital assays with single-molecule resolution.

Catalytic Nucleic Acids: Biochemistry, Chemical Biology, Biosensors, and Nanotechnology.

Since the initial discovery of ribozymes in the early 1980s, catalytic nucleic acids have been used in different areas. Compared with protein enzymes, catalytic nucleic acids are programmable in structure, easy to modify, and more stable especially for DNA. We take a historic view to summarize a few main interdisciplinary areas of research on nucleic acid enzymes that may have broader impacts. Early efforts on ribozymes in the 1980s have broken the notion that all enzymes are proteins, supplying new evidence for the RNA world hypothesis. In 1994, the first catalytic DNA (DNAzyme) was reported. Since 2000, the biosensor applications of DNAzymes have emerged and DNAzymes are particularly useful for detecting metal ions, a challenging task for enzymes and antibodies. Combined with nanotechnology, DNAzymes are key building elements for switches allowing dynamic control of materials assembly. The search for new DNAzymes and ribozymes is facilitated by developments in DNA sequencing and computational algorithms, further broadening our fundamental understanding of their biochemistry.

Cationic copolymer enhances 8–17 DNAzyme and MNAzyme activities

DNAzymes are DNA molecules capable of catalytic activity. The catalytic core of DNAzymes can be separated and conjugated with target binding arms to create allosteric DNAzymes known as multi-component nucleic acid enzymes (MNAzymes). Two widely used DNAzymes are the 10-23 and the 8-17 DNAzymes. These DNAzymes differ in catalytic core structures, cleavage sites, and reactive metal ion cofactors. Previously we showed that the presence of a cationic comb-type polymer poly(l-lysine)-graft-dextran (PLL-g-Dex) improved activities of the 10-23 DNAzyme and MNAzyme by facilitating assembly of the catalytic complex. In this work, we demonstrate that PLL-g-Dex enhances activities of the 8-17 DNAzyme and MNAzyme; poly(allylamine)-graft-dextran and cationic homopolymers did not enhance activities. Metal ion and pH dependences were observed in the presence of PLL-g-Dex, suggesting that the cationic copolymer did not impede the interaction between the metal ion and the DNA-based enzymes. Thus, PLL-g-Dex has chaperone-like activity for DNAzymes and MNAzymes regardless of structures, cleavage sites, and cofactors.

Nucleic Acid Catalysis under Potential Prebiotic Conditions.

Catalysis by nucleic acids is indispensable for extant cellular life, and it is widely accepted that nucleic acid enzymes were crucial for the emergence of primitive life 3.5‐4 billion years ago. However, geochemical conditions on early Earth must have differed greatly from the constant internal milieus of today's cells. In order to explore plausible scenarios for early molecular evolution, it is therefore essential to understand how different physicochemical parameters, such as temperature, pH, and ionic composition, influence nucleic acid catalysis and to explore to what extent nucleic acid enzymes can adapt to non‐physiological conditions. In this article, we give an overview of the research on catalysis of nucleic acids, in particular catalytic RNAs (ribozymes) and DNAs (deoxyribozymes), under extreme and/or unusual conditions that may relate to prebiotic environments.

The L-platform/L-scaffold framework: a blueprint for RNA-cleaving nucleic acid enzyme design.

We develop an L-platform/L-scaffold framework we hypothesize may serve as a blueprint to facilitate site-specific RNA-cleaving nucleic acid enzyme design. Building on the L-platform motif originally described by Suslov and coworkers, we identify new critical scaffolding elements required to anchor a conserved general base guanine (“L-anchor”) and bind functionally important metal ions at the active site (“L-pocket”). Molecular simulations, together with a broad range of experimental structural and functional data, connect the L-platform/L-scaffold elements to necessary and sufficient conditions for catalytic activity. We demonstrate that the L-platform/L-scaffold framework is common to five of the nine currently known naturally occurring ribozyme classes (Twr, HPr, VSr, HHr, Psr), and intriguingly from a design perspective, the framework also appears in an artificially engineered DNAzyme (8–17dz). The flexibility of the L-platform/L-scaffold framework is illustrated on these systems, highlighting modularity and trends in the variety of known general acid moieties that are supported. These trends give rise to two distinct catalytic paradigms, building on the classifications proposed by Wilson and coworkers and named for the implicated general base and acid. The “G + A” paradigm (Twr, HPr, VSr) exclusively utilizes nucleobase residues for chemistry, and the “G + M + ” paradigm (HHr, 8–17dz, Psr) involves structuring of the “L-pocket” metal ion binding site for recruitment of a divalent metal ion that plays an active role in the chemical steps of the reaction. Finally, the modularity of the L-platform/L-scaffold framework is illustrated in the VS ribozyme where the “L-pocket” assumes the functional role of the “L-anchor” element, highlighting a distinct mechanism, but one that is functionally linked with the hammerhead ribozyme.

Target-Catalyzed Self-Growing Spherical Nucleic Acid Enzyme (SNAzyme) as a Double Amplifier for Ultrasensitive Chemiluminescence MicroRNA Detection.

Portable chemiluminescence (CL) imaging with a smartphone has shown a great promise for point-of-care testing of diseases, especially for acute myocardial infarction (AMI), which may occur abruptly. A challenge remains how to improve the imaging sensitivity that usually is several orders of magnitude lower than those of counterpart methodologies using the sophisticated equipment. Toward this goal, here, we report the target-triggered in situ growth of AuNP@hairpin-DNA nanoprobes into spherical nucleic acid enzymes (SNAzymes), which serve as both nanolabels and amplifiers for portable CL imaging of microRNAs (miRNAs) with an ultrahigh sensitivity comparable to that of the instrumental measurement under same conditions. A G-quadruplex (G4) DNA dense layer is dynamically produced on the gold nanocore via a DNAzyme machine-driven hairpin cleaving and captures the cofactor hemin to form the SNAzymes with higher peroxidase activity and stronger nuclease resistance than the commonly used G4 DNAzymes. The matured SNAzymes are then utilized as catalytic labels in a luminol-artesunate CL system for miRNA imaging with a smartphone as the portable detector. In this way, two AMI-related miRNAs, miRNA-499 and miRNA-133a, are successfully detected in real patients' serum with a naked eye-visualized CL change at 10 fM, showing a 5 order of magnitude improvement on the sensitivity of visualizing the same disease markers in clinical circulating blood as compared to the reported strategy. In addition, a good selectivity of our developed CL imaging platform is demonstrated. These unique features make it promising to employ this portable imaging platform for clinical AMI diagnosis.

Biosensors Made of Synthetic Functional Nucleic Acids Toward Better Human Health.

The invention of the in vitro selection technique, or SELEX (Systematic Evolution of Ligands by EXponential enrichment), in the early 1990s, identified single-stranded (ss) nucleic acid molecules with catalytic or binding properties from random-sequence nucleic acid libraries.1–3 This technique has given rise to intensive examinations of synthetic functional nucleic acids (aptamers and nucleic acid enzymes) for wide-ranging applications including biosensors, diagnostics and therapeutics.4–9After nearly three decades of research by many groups around the world, many functional nucleic acids have been isolated and characterized. A few well-known examples are provided in Figure 1, including two RNA-cleaving DNAzymes (8-17 and 10-23),10 a peroxidase-mimicking DNAzyme (PS2.M),11 two DNA aptamers that bind human α-thrombin (TBA15 and TBA29),12,13 a DNA aptamer that binds ATP,14 and a modified nucleic acid aptamer that binds human VEGF-165 (Pegaptanib).15 Thousands of papers have been published describing the us...

Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection

Multi-component nucleic acid enzymes (MNAzymes) are allosteric deoxyribozymes that are activated upon binding of a specific nucleic acid effector. MNAzyme activity is limited due to an insufficient assembly of the MNAzyme and its turnover. In this work, we describe the successful improvement of MNAzyme reactivity and selectivity by addition of cationic copolymers, which exhibit nucleic acid chaperone-like activity. The copolymer allowed a 210-fold increase in signal activity and a 95-fold increase in the signal-to-background selectivity of MNAzymes constructed for microRNA (miRNA) detection. The selectivity of the MNAzyme for homologous miRNAs was demonstrated in a multiplex format in which isothermal reactions of two different MNAzymes were performed. In addition, the copolymer permitted miRNA detections even in the presence of a ribonuclease which is ubiquitous in environments, indicating the protective effect of the copolymer against ribonucleases.

Target-Induced Payload Amplification for Spherical Nucleic Acid Enzyme (SNAzyme)-Catalyzed Electrochemiluminescence Detection of Circulating microRNAs.

A high level of circulating myocardial microRNAs (miRNAs) is commonly considered as one indicator of acute myocardial infarction (AMI) for early stage diagnosis, and hence, it is of particular significance to develop ultrasensitive methodologies for detecting these miRNAs in circulating blood. Here we build an electrochemiluminescence (ECL) sensing platform for circulating miRNAs utilizing AuNPs@G-quadruplex (G4) spherical nucleic acid enzyme (SNAzyme) as the nanocatalyst, which shows good stability, strong nuclease resistance, and improved catalytic performance toward a luminol-H2O2 ECL system than the commonly used G4 DNAzyme. Target miRNA is employed to open the probe hairpin DNA to trigger the cascade amplification and then produce a long dsDNA chain with many sticky linkers that capture the SNAzyme nanocatalyst onto the electrode. In this way, two AMI-related miRNAs are detected accurately. The detection limit is 0.4 fM (S/N = 3), and the platform shows high selectivity in circulating blood. Given the good controllability, our ECL biosensors are promising for developing clinical diagnostic platforms for multiple indicators.

Cellular Concentrations of Nucleotide Diphosphate-Chelated Magnesium Ions Accelerate Catalysis by RNA and DNA Enzymes.

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg2+free. We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

Multi-level patterning nucleic acid photolithography

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage.

Subzymes: Regulating DNAzymes for point of care nucleic acid sensing

Rapid, sensitive and affordable nucleic acid sensors that can operate in a point of care (POC) setting are highly desired; however, their availability and implementation remain limited. We report the development of a strategy for building sensors which use novel catalytic nucleic acid structures, herein referred to as ‘Subzymes’. We examine the effectiveness of Subzymes to mediate signal amplification in a universal manner allowing faster rates of detection. We created Subzymes by manufacturing composite oligonucleotides that contain a catalytic nucleic acid component and a substrate component. We demonstrate that the activity of some catalytic DNAzyme components can be inhibited by attaching the Subzymes to micro-particles. Subsequent cleavage of a Subzyme’s internal substrate results in the release and activation of the surface-bound DNAzyme, thus providing a mechanism to control the catalytic activity of the DNAzyme. We demonstrate that released DNAzymes are capable of cleaving fluorescent-labelled reporter substrates to generate a signal, thus confirming the restoration of their catalytic activity. The addition of Subzymes to reactions where the detection of a target is achieved by multi-component nucleic acid enzymes, known as PlexZymes, showed improved target sensitivities in a shorter amount of time (10 pM of target detected in under 60 min), demonstrating rapid detection of nucleic acid targets without the use of protein-enzymes. Subzymes are constructed from low-cost materials and operate under isothermal reaction conditions which are advantageous for on-site diagnostic testing. Thus, Subzymes offer a universal, rapid and affordable tool for nucleic acid sensing, providing new avenues for POC testing.