DNA-Templated Click Ligation Chain Reaction Catalyzed by Heterogeneous Cu2O for Enzyme-Free Amplification and Ultrasensitive Detection of Nucleic Acids.

Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via DNA-templated click ligation chain reaction (DT-CLCR) catalyzed by a heterogeneous nanocatalyst made of Cu2O (hnCu2O). This hnCu2O-DT-CLCR method is built on two cross-amplifying hnCu2O-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCu2O-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCu2O-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCu2O-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCu2O-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications.

Systematic evolution of ligands by exponential enrichment (SELEX) has been used to discover thousands of aptamers since its development in 1990. Aptamers are short single-stranded oligonucleotides capable of binding to targets with high specificity and selectivity through structural recognition. While aptamers offer advantages over other molecular recognition elements such as their ease of production, smaller size, extended shelf-life, and lower immunogenicity, they have yet to show significant success in real-world applications. By analyzing the importance of structured library designs, reviewing different SELEX methodologies, and the effects of chemical modifications, we provide a comprehensive overview on the production of aptamers for applications in drug delivery systems, therapeutics, diagnostics, and molecular imaging.

High-Precision Viral Detection Using Electrochemical Kinetic Profiling of Aptamer-Antigen Recognition in Clinical Samples and Machine Learning.

High-precision viral detection at point of need with clinical samples plays a pivotal role in the diagnosis of infectious diseases and the control of a global pandemic. However, the complexity of clinical samples that often contain very low viral concentrations makes it a huge challenge to develop simple diagnostic devices that do not require any sample processing and yet are capable of meeting performance metrics such as very high sensitivity and specificity. Herein we describe a new single-pot and single-step electrochemical method that uses real-time kinetic profiling of the interaction between a high-affinity aptamer and an antigen on a viral surface. This method generates many data points per sample, which when combined with machine learning, can deliver highly accurate test results in a short testing time. We demonstrate this concept using both SARS-CoV-2 and Influenza A viruses as model viruses with specifically engineered high-affinity aptamers. Utilizing this technique to diagnose COVID-19 with 37 real human saliva samples results in a sensitivity and specificity of both 100 % (27 true negatives and 10 true positives, with 0 false negative and 0 false positive), which showcases the superb diagnostic precision of this method.

Engineering a Ligase Binding DNA Aptamer into a Templating DNA Scaffold to Guide the Selective Synthesis of Circular DNAzymes and DNA Aptamers.

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.

Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing.

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.

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.

Detection of Large Genomic RNA via DNAzyme-Mediated RNA Cleavage and Rolling Circle Amplification: SARS-CoV-2 as a Model.

A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h.

Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library.

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.

Functional nucleic acid biosensors utilizing rolling circle amplification.

Functional nucleic acids (FNAs), including DNA aptamers and DNAzymes, are finding increasing use as molecular recognition elements for point-of-care (POC) assays and sensors. An ongoing challenge in the development of FNA-based POC sensors is the ability to achieve detection of low levels of analyte without compromising assay time and ease of use. Rolling circle amplification (RCA) is a leading nucleic acid (NA) isothermal amplification method which can be coupled with FNAs for the ultrasensitive detection of non-NA targets. Herein we examine the key considerations required when designing FNA-coupled biosensors utilizing RCA. Specifically, we describe methods for using FNAs as inputs to regulate RCA, various modes of RCA amplification, and methods to detect the output of the RCA reaction, along with how these can be combined to allow detection of non-NA targets. Recent progress on development of portable optical and electrochemical POC devices that incorporate RCA is then described, followed by a summary of key challenges and opportunities in the field.

A Fluorogenic DNAzyme for A Thermally Stable Protein Biomarker from Fusobacterium nucleatum, a Human Bacterial Pathogen.

Fusobacterium nucleatum has been correlated to many poor human conditions including oral infections, adverse pregnancies and cancer, and thus molecular tools capable of detecting this human pathogen can be used to develop diagnostic tests for them. Using a new selection method targeting thermally stable proteins without a counter-selection step, we derived an fluorogenic RNA-cleaving DNAzyme, named RFD-FN1, that can be activated by a thermally stable protein target that is unique to F. nucleatum subspecies. High thermal stability of protein targets is a very desirable attribute for DNAzyme-based biosensing directly with biological samples because nucleases found inherently in these samples can be heat-inactivated. We further demonstrate that RFD-FN1 can function as a fluorescent sensor in both human saliva and human stool samples. The discovery of RFD-FN1 paired with a highly thermal stable protein target presents opportunities for developing simpler diagnostic tests for this important pathogen.

A Simple Colorimetric Au-on-Au Tip Sensor with a New Functional Nucleic Acid Probe for Food-borne Pathogen Salmonella typhimurium.

An Au-on-Au tip sensor is developed for the detection of Salmonella typhimurium (Salmonella), using a new synthetic nucleic acid probe (NAP) as a linker for the immobilization of a DNA-conjugated Au nanoparticle (AuNP) onto a DNA-attached thin Au layer inside a pipette tip. In the presence of Salmonella, RNase H2 from Salmonella (STH2) cleaves the NAP and the freed DNA-conjugated AuNP can be visually detected by a paper strip. This portable biosensor does not require any electronic, electrochemical or optical equipment. It delivers a detection limit of 3.2×103  CFU mL-1 for Salmonella in 1 h without cell-culturing or signal amplification and does not show cross-reactivity with several control bacteria. Further, the sensor reliably detects Salmonella spiked in food samples, such as ground beef and chicken, milk, and eggs. The sensor can be reused and is stable at ambient temperature, showing its potential as a point-of-need device for the prevention of food poisoning by Salmonella.

A Colorimetric Biosensing Platform with Aptamers, Rolling Circle Amplification and Urease-Mediated Litmus Test.

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.

An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function.

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.

Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

Target-Mediated 5'-Exonuclease Digestion of DNA Aptamers with RecJ to Modulate Rolling Circle Amplification for Biosensing.

We report a new method for biosensing based on the target-mediated resistance of DNA aptamers against 5'-exonuclease digestion, allowing them to act as primers for rolling circle amplification (RCA). A target-bound DNA strand containing an aptamer region on the 5'-end and a primer region on the 3'-end is protected from 5'-exonuclease digestion by RecJ exonuclease in a target-dependent manner. As the protected aptamer is at the 5'-end, the exposed primer on the 3'-end can participate in RCA in the presence of a circular template to generate a turn-on sensor. Without target, RecJ digests the primer and prevents RCA from occurring, allowing quantitative fluorescence detection of both thrombin, a protein, and ochratoxin A (OTA), a small molecule, at picomolar concentrations.

Functional Nucleic Acids for Pathogenic Bacteria Detection.

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.

Fluorescent property of carbon dots extracted from cigarette smoke and the application in bio-imaging.

Cigarette smoke is one of the six major pollution sources in the room air. It contains large number of particles with size less than 10 nm. There exist carbon dots (CDs) in cigarette smoke which have strong fluorescence and with good bio-compatibility and low toxicity. CDs in cigarette smoke can be applied in bio-imaging which has great potential applications in the integration of cancer diagnosis and treatment. In this paper, CDs were extracted from cigarette smoke. Then, sodium borohydride was added to CDs aqueous solution for reduction and the reduced CDs (R-CDs) were used for biological cell imaging. The results indicate that the CDs with the particle size <10 nm in cigarette smoke are self-assembled by the polymerizated polycyclic aromatic hydrocarbons (PAHs) and ammonium nitrite which are disk nano-structure composed of sp2/sp3 carbon and oxygen/nitrogen groups or polymers. Sodium borohydride can reduce the carbonyl group on the surface of CDs to hydroxyl group and increase the ratio of the Na 1s ratio of the CDs from 1.86 to 7.42. The CDs can emit blue fluorescence under ultraviolet irradiation. After reduction, the R-CDS have the intensity of fluorescence 7.2 times than before and the fluorescence quantum yield increase from 6.13% to 8.86%. The photoluminescence (PL) wavelength of R-CDS have red-shift of 7 nm which was due to the increasing of Na element ratio. The onion epidermal cells labeled with R-CDs show that the CDs could pass through the cell wall into the cell and reach the nucleus. The cell wall and the nucleus could be clearly visualized. CDs also shows low toxicity to human bronchial epithelial cells (BEAS-2B) with good biological activity. The obtained results indicate that the CDs and R-CDs have good fluorescent property which could be used as bio-imaging agent.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

Invited for the cover of this issue are John Brennan, Yingfu Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

Evolution of a highly functional circular DNA aptamer in serum.

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.