Novel fluorescence nano-orbital biosensor for highly sensitive microRNA detection.

BACKGROUND: MicroRNAs play an important role in regulating cell function and gene expression. Early prevention and clinical diagnosis of diseases have high requirements for high-sensitivity detection of microRNAs. Due to the limitations of tedious operation and large sample size, miRNA with small molecular weight and low expression abundance cannot be accurately detected in traditional miRNA detection. To improve the sensitivity and accuracy of detection, we established a novel biosensor based on nucleic acid circuit of signal amplification, which converted miRNA recognition into a fluorescence signal for amplification. RESULTS: We designed a biosensor based on an exponential amplification reaction with cascaded HCR and DNAzyme nucleic acid circuit (named E-NOF biosensor) by amplicon sub-fragments to trigger the construction of fluorescence nano-orbitals (NOF), which could be used to detect miRNA ultrasensitively. By modifying two fluorophores (Cy3 and Cy5) on the chain of constructing nano-orbitals, when the amplicon triggered the construction of nano-orbitals, fluorescence resonance energy transfer (FRET) occurred between Cy3 and Cy5, and then two fluorescence signals with different trends could be observed. Therefore, through the ratio of the two signals, we could quantitatively and quickly detect the miRNA from 1 fM to 100 nM, and the E-NOF biosensor detection limit was as low as 0.129 fM. Furthermore, the HCR nucleic acid circuit cascaded with DNAzyme could enrich the fluorophores on the nano-orbitals and significantly enhance the fluorescence signal by accelerating the reaction rate. SIGNIFICANCE: According to our understanding, the E-NOF biosensor is the first strategy to cascade EXPAR with HCR and DNAzyme nucleic acid circuit for miRNA-1246 detection. Accurate results can be obtained in only 120 min. Compared with the traditional HCR system, the sensitivity of the new E-NOF biosensor is increased by 1 × 109 times. Furthermore, the biosensor can also detect biomarkers in human serum samples. It has great potential in miRNA detection and identification.

Structure of a 10-23 Deoxyribozyme Exhibiting a Homodimer Conformation.

Deoxyribozymes (DNAzymes) are in vitro evolved DNA sequences capable of catalyzing chemical reactions. The RNA cleaving 10-23 DNAzyme was the first DNAzyme to be evolved and possesses clinical and biotechnical applications as a biosensor and a knockdown agent. DNAzymes do not require the recruitment of other components to cleave RNA and can turnover, thus they have a distinct advantage over other knockdown methods (siRNA, CRISPR, morpholinos). Despite this, a lack of structural and mechanistic information has hindered the optimization and application of the 10-23 DNAzyme. Here, we report a 2.7 Å crystal structure of the RNA cleaving 10-23 DNAzyme in a homodimer conformation. Although proper coordination of the DNAzyme to substrate is observed along with intriguing patterns of bound magnesium ions, the dimer conformation likely does not capture the true catalytic form of the 10-23 DNAzyme.

Allosteric Nucleic Acid Enzyme: A Versatile Stimuli-Responsive Tool for Molecular Computing and Biosensing Nanodevices.

Allostery is a naturally occurring mechanism in which effector binding induces the modulation and fine control of a related biomolecule function. Deoxyribozyme (DNAzyme) with catalytic activity and substrate recognition ability is ideal to be regulated by allosteric strategies. However, the current regulations frequently confront various obstacles, such as severe activity decay, signal leakage, and limited effectors. In this work, a rational regulation strategy for developing versatile effectors-responsive allosteric nucleic acid enzyme (ANAzyme) by introducing an allosteric domain in response to diverse effectors is established. The enzyme-like activity of this re-engineered ANAzyme can be modulated in a more predictable and fine way compared with the previous DNAzyme regulation strategies. Based on the allosteric strategy, the construction of allosterically coregulatory nanodevices and a series of basic logic gates and logic circuits are achieved, demonstrating that the proposed ANAzyme-regulated strategy showed great potential in molecular computing. Given these facts, the rational design of ANAzyme with the allosteric domain presented here can expand the available toolbox to develop a variety of stimuli-responsive allosteric DNA materials, including molecular machines, computing systems, biosensing platforms, and gene-silencing tools.

A Novel DNAzyme-Based Fluorescent Biosensor for Detection of RNA-Containing Nipah Henipavirus.

Nipah virus (NiV) is a zoonotic RNA virus which infects humans and animals in Asian countries. Infection in humans occurs in different forms, from asymptomatic infection to fatal encephalitis, and death occurred in 40-70% of those infected in outbreaks that occurred between 1998 and 2018. Modern diagnostics is carried out by real-time PCR to identify pathogens or by ELISA to detect antibodies. Both technologies are labor-intensive and require the use of expensive stationary equipment. Thus, there is a need to develop alternative simple, fast and accurate test systems for virus detection. The aim of this study was to develop a highly specific and easily standardized system for the detection of Nipah virus RNA. In our work, we have developed a design for a Dz_NiV biosensor based on a split catalytic core of deoxyribozyme 10-23. It was shown that the assembly of active 10-23 DNAzymes occurred only in the presence of synthetic target Nipah virus RNA and that this was accompanied by stable fluorescence signals from the cleaved fluorescent substrates. This process was realized at 37 °C, pH 7.5, and in the presence of magnesium ions, with a 10 nM limit of detection achieved for the synthetic target RNA. Constructed via a simple and easily modifiable process, our biosensor may be used for the detection of other RNA viruses.

Functional Zeolitic Imidazolate Framework for Robust l-Deoxyribozyme-Based Therapy.

Programmable chiral biocatalysis represents a promising therapeutic strategy for its high stereospecific control over various biotransformations (e.g., chiral Aß isomerization) of living entities yet is rarely explored. With an extraordinary resistance to nuclease digestion, the non-natural left-handed deoxyribozyme (l-DNAzyme) therapy is constrained by inefficient delivery/release and insufficient cofactors supply. Herein, an efficient adenosine triphosphate (ATP)-stimulated disassembly of l-histidine (l-His)-integrated ZIF-8 (l-His-ZIF-8) is reported for sustaining the l-DNAzyme-amplified photodynamic therapy. This self-sufficient l-therapeutic platform can intelligently release the l-DNAzyme probe and simultaneously supply l-His DNAzyme cofactors via endogenous ATP. Then, the intrinsic microRNA-21 catalyzes the generation of robust l-DNAzyme via the catalytic hybridization reaction for activating the photosensitizer with multiplied guaranteed therapeutic operation. This l-therapeutic strategy opens up great prospects for more precise diagnosis and customized gene silencing-based therapy.

Evolution of Proliferative Model Protocells Highly Responsive to the Environment.

In this review, we discuss various methods of reproducing life dynamics using a constructive approach. An increase in the structural complexity of a model protocell is accompanied by an increase in the stage of reproduction of a compartment (giant vesicle; GV) from simple reproduction to linked reproduction with the replication of information molecules (DNA), and eventually to recursive proliferation of a model protocell. An encounter between a plural protic catalyst (C) and DNA within a GV membrane containing a plural cationic lipid (V) spontaneously forms a supramolecular catalyst (C@DNA) that catalyzes the production of cationic membrane lipid V. The local formation of V causes budding deformation of the GV and equivolume divisions. The length of the DNA strand influences the frequency of proliferation, associated with the emergence of a primitive information flow that induces phenotypic plasticity in response to environmental conditions. A predominant protocell appears from the competitive proliferation of protocells containing DNA with different strand lengths, leading to an evolvable model protocell. Recently, peptides of amino acid thioesters have been used to construct peptide droplets through liquid-liquid phase separation. These droplets grew, owing to the supply of nutrients, and were divided repeatedly under a physical stimulus. This proposed chemical system demonstrates a new perspective of the origins of membraneless protocells, i.e., the "droplet world" hypothesis. Proliferative model protocells can be regarded as autonomous supramolecular machines. This concept of this review may open new horizons of "evolution" for intelligent supramolecular machines and robotics.

Toward a Home Test for COVID-19 Diagnosis: DNA Machine for Amplification-Free SARS-CoV-2 Detection in Clinical Samples.

Nucleic acid-based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein-enzyme-free nano-construction, named four-armed DNA machine (4DNM), that requires neither an amplification stage nor a high-temperature annealing step for SARS-CoV-2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA-binding arms. Additional arms were found to improve the limit of detection at least 10-fold. The sensor distinguished SARS-CoV-2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT-qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point-of-care or home diagnostic test.

Pushing the Limits of Nucleic Acid Function.

For many decades it was thought that information storage and information transfer were the main functions of nucleic acids. However, artificial evolution experiments have shown that the functional potential of DNA and RNA is much greater. Here I provide an overview of this technique and highlight recent advances which have increased its potency. I also describe how artificial evolution has been used to identify nucleic acids with extreme functions. These include deoxyribozymes that generate unusual products such as light, tiny motifs made up of fewer than ten nucleotides, ribozymes that catalyze complex reactions such as RNA polymerization, information-rich sequences that encode overlapping ribozymes, motifs that catalyze reactions at rates too fast to be followed by manual pipetting, and functional nucleic acids which are active in extreme conditions. Such motifs highlight the limits of our knowledge and provide clues about as of yet undiscovered functions of DNA and RNA.

Optimizing the Chemiluminescence of a Light-Producing Deoxyribozyme.

Supernova is a chemiluminescent deoxyribozyme recently discovered in our group. It transfers the phosphate group from the 1,2-dioxetane substrate CDP-Star to its 5' hydroxyl group, which triggers a decomposition reaction and the production of light. Here we investigated the effects of reaction conditions on the ability of Supernova to generate a chemiluminescent signal (using a plate reader assay) and to phosphorylate itself (using a ligation assay). Our experiments indicate that multiple zinc ions are required for catalytic function, suggesting links between Supernova and protein enzymes that catalyze similar reactions. They also show how factors such as pH, potassium concentration, CDP-Star concentration, and DNA concentration affect the reaction. By combining information from different experiments, the rate enhancement of light production was increased by more than 1000-fold. These results should be useful for applications in which Supernova is used as a sensor.

Obtaining Crystals of Nucleic Acids in Complex with the Protein U1A Using the Soaking Method.

X-ray crystallography is one of the most prominent techniques for determining high-resolution structures of nucleic acids. The major challenges are to obtain well-diffracting single crystals and to solve the phase problem. The absence of structural information impedes the elucidation of the molecular details of biological processes. A particularly intriguing example is the RNA-cleavage catalyzed by the 10-23 deoxyribozyme (DNAzyme). This DNAzyme consists of a catalytic core that is flanked by two substrate binding arms, which can be designed to bind any RNA of interest. Structure elucidation of the 10-23 DNAzyme in a biologically relevant conformation faces three major challenges: (1) stabilization of the RNA substrate to capture the DNA:RNA complex in the pre-catalytic conformation, (2) prevention of the formation of an artificial duplex conformation due to a self-complementary sequence in the catalytic core of the DNAzyme, and (3) the crystallization of nucleic acids with their uniform surfaces. Here, we provide a protocol for an innovative strategy facilitating the crystallization of protein:nucleic acid complexes using a soaking approach and discuss on how to apply this protocol for the structure elucidation of the 10-23 DNAzyme. For this purpose, we describe the purification procedure of an optimized variant of the RNA-binding protein U1A, the crystallization of this specific U1A variant, the soaking process with its specific RNA hairpin loop, and finally suggest a strategy for applying this procedure on the 10-23 DNAzyme in complex with its specific RNA target.

Generation of RNA with 2', 3'-Cyclic Phosphates by Deoxyribozyme Cleavage in Frozen Solutions.

The generation of terminal 2', 3'-cyclic phosphates on RNA oligomers is an important process in the study of tRNA splicing and repair, ribozyme catalysis, and RNA circularization. Here, we describe a simple method for producing 2', 3'-cyclic phosphate functionalized RNA by the deoxyribozyme-catalyzed cleavage of a short 3'-RNA overhang in frozen solution. This method avoids the nonspecific modification and degradation of RNA and attached functional groups (e.g., fluorophores) inherent in other methods, and the use of frozen conditions enables cleavage at very low divalent metal ion concentrations, limiting RNA hydrolysis.

Supernova: A Deoxyribozyme that Catalyzes a Chemiluminescent Reaction.

Functional DNA molecules are useful components in nanotechnology and synthetic biology. To expand the toolkit of functional DNA parts, in this study we used artificial evolution to identify a glowing deoxyribozyme called Supernova. This deoxyribozyme transfers a phosphate from a 1,2-dioxetane substrate to its 5' hydroxyl group, which triggers a chemiluminescent reaction and a flash of blue light. An engineered version of Supernova is only catalytically active in the presence of an oligonucleotide complementary to its 3' end, demonstrating that light production can be coupled to ligand binding. We anticipate that Supernova will be useful in a wide variety of applications, including as a signaling component in allosterically regulated sensors and in logic gates of molecular computers.

Complexing deoxyribozymes with RNA aptamers for detection of the small molecule theophylline.

Biointegrative information processing systems offer a great advantage to autonomous biodevices, as their capacity for biological computation provides the ability to sense the state of more complex environments and better integrate with downstream biological regulation systems. Deoxyribozymes (DNAzymes) and aptamers are of interest to such computational biosensing systems due to the enzymatic properties of DNAzymes and the ligand-inducible conformational structures of aptamers. Herein, we describe a novel method for providing ligand-responsive allosteric control to a DNAzyme using an RNA aptamer. We designed a NOT-logic-compliant E6 DNAzyme to be complementary to an RNA aptamer targeting theophylline, such that the aptamer competitively interacted with either theophylline or the DNAzyme, and disabled the DNAzyme only when theophylline concentration was below a given threshold. Out of our seven designed "complexing aptazymes," three demonstrated effective theophylline-responsive allosteric regulation (2.84 ± 3.75%, 4.97 ± 2.92%, and 8.91 ± 4.19% activity in the absence of theophylline; 46.29 ± 3.36%, 50.70 ± 10.15%, and 61.26 ± 6.18% activity in the presence of theophylline). Moreover, the same three complexing aptazymes also demonstrated the ability to semi-quantitatively determine the concentration of theophylline present in solution, successfully discriminating between therapeutically ineffective (<20 µM), safe (20-100 µM), and toxic (>100 µM) theophylline concentrations. Our method of using an RNA aptamer for ligand-responsive allosteric control of a DNAzyme expands the way aptamers can be configured for biosensing, and suggests a pathway for embedding DNAzymes to provide enhanced information processing and control of biological systems.

Colocalization Analysis of Lipo-Deoxyribozyme Consisting of DNA and Protic Catalysts in a Vesicle-Based Protocellular Membrane Investigated by Confocal Microscopy.

The linkage between the self-reproduction of compartments and the replication of DNA in a compartment is a crucial requirement for cellular life. In our giant vesicle (GV)-based model protocell, this linkage is achieved through the action of a supramolecular catalyst composed of membrane-intruded DNA and amphiphilic acid catalysts (C@DNA) in a GV membrane. In this study, we examined colocalization analysis for the formation of the supramolecular catalyst using a confocal laser scanning fluorescence microscope with high sensitivity and resolution. Red fluorescence spots emitted from DNA tagged with Texas Red (Texas Red-DNA) were observed in a GV membrane stained with phospholipid tagged with BODIPY (BODIPY-HPC). To our knowledge, this is the first direct observation of DNA embedded in a GV-based model protocellular membrane containing cationic lipids. Colocalization analysis based on a histogram of frequencies of "normalized mean deviation product" revealed that the frequencies of positively correlated [lipophilic catalyst tagged with BODIPY (BODIPY-C) and Texas Red-DNA] were significantly higher than those of [BODIPY-HPC and Texas Red-DNA]. This result demonstrates the spontaneous formation of C@DNA in the GV membrane, which serves as a lipo-deoxyribozyme for producing membrane lipids from its precursor.

MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy.

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA-MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

Secondary Structure Libraries for Artificial Evolution Experiments.

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.

Structural and functional characterization of complexes between heme and dimeric parallel G-quadruplex DNAs.

Heme has been receiving considerable interest as a prosthetic group of ribozymes and deoxyribozymes, because heme-bound nucleic acids exhibit peroxidase-like catalytic activities (Travascio, P., Li, Y., and Sen, D. (1998) Chem. Biol, 5, 505-517). The interaction of heme with dimeric G-quadruplexes formed from d(TAGGGTTAGGGT) and d(TAGGGTTAGGGA) has been characterized to gain a deeper understanding of the molecular recognition of G-quadruplex DNAs by heme. We found that heme binds selectively to the 3'-terminal G-quartet of a dimeric parallel G-quadruplex of d(TAGGGTTAGGGT), whereas binding of heme to a dimeric antiparallel G-quadruplex of d(TAGGGTTAGGGA) does not occur, suggesting that an orderly arrangement of the constituent guanine deoxyribose rings, with respect to the G-quartet plane, is crucial for the binding of heme to the DNA. The preferential binding of heme to the 3'-terminal G-quartet of parallel G-quadruplex DNAs allowed a systematic modification of the heme environment in the complex through the DNA sequence. The activity of the complexes was found to increase with increasing number of adenine bases adjacent to the heme in the complexes, possibly due to improvement of the accessibility of aromatic substrate, i.e., 10-acetyl-3,7-dihydroxyphenoxazine, to the heme, and an increase in the frequency of appearance of a specific orientation of the adenine bases, with respect to the heme, optimized for its activity as an acid-base catalyst to enhance the peroxidase activity of the complex.

DNAzymes as key components of biosensing systems for the detection of biological targets.

DNAzymes are synthetic functional nucleic acids that have found widespread use in biosensing applications both for molecular recognition and signal generation. Two classes of DNAzymes have proved particularly effective for use in proof-of-concept biosensing systems, namely RNA-cleaving DNAzymes (RCDs) and peroxidase mimicking DNAzymes (PMDs). RCDs catalyze the site-specific cleavage reaction of an RNA dinucleotide junction, generating two cleavage fragments. PMDs are capable of catalyzing peroxidation reactions of chromophores, thereby generating a measurable signal. Herein, we review the use of these DNAzymes in biomedical assays and diagnostics, and show that this emerging field should have great promise for biosensor development over the next few decades.

Characterization and Optimization of a Deoxyribozyme with a Short Left Binding Arm.

Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have broad applications in biotechnology. In vitro selected RNA-cleaving deoxyribozymes normally contain two substrate-binding arms and a central catalytic core region. Here, we describe the systematic characterization and optimization of an RNA-cleaving deoxyribozyme with an unusually short left binding arm, and its special sequence requirement for its optimal catalytic activity.

Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures.

Molecular analysis of RNA through hybridization with sequence-specific probes is challenging due to the intrinsic ability of RNA molecules to form stable secondary and tertiary structures. To overcome the energy barrier toward the probe-RNA complex formation, the probes are made of artificial nucleotides, which are more expensive than their natural counterparts and may still be inefficient. Here, we propose the use of a multicomponent probe based on an RNA-cleaving deoxyribozyme for the analysis of highly structured RNA targets. Efficient interrogation of two native RNA from Saccharomyces cerevisiae-a transfer RNA (tRNA) and 18S ribosomal RNA (rRNA)-was achieved at ambient temperature. We achieved detection limits of tRNA down to ∼0.3 nM, which is two orders of magnitude lower than that previously reported for molecular beacon probes. Importantly, no probe annealing to the target was required, with the hybridization assay performed at 37°C. Excess of nonspecific targets did not compromise the performance of the probe, and high interrogation efficiency was maintained by the probes even in complex matrices, such as cell lysate. A linear dynamic range of 0.3-150 nM tRNA was demonstrated. The probe can be adapted for differentiation of a single mismatch in the tRNA-probe complex. Therefore, this study opens a venue toward highly selective, sensitive, robust, and inexpensive assays for the interrogation of biological RNA.