Making target sites in large structured RNAs accessible to RNA-cleaving DNAzymes through hybridization with synthetic DNA oligonucleotides.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary.

Protein-Templated Click Ligation Reaction Triggered by Protein-Split Aptamer Interactions.

DNA-templated reactions have found wide applications in sensing and drug discovery. However, this strategy has been limited to the use of nucleic acids as templating elements to direct the proximity effect. Herein, we describe a versatile protein-templated split aptamer click ligation reaction (PT-SpA-CLR) in which the protein template-induced covalent proximity ligation of split aptamer elements enables translating protein/aptamer binding events into the output of ligated DNA products. A ligation yield of >80% is observed for three model protein templates, including VEGF165, PDGF-BB, and SARS-CoV-2 S1. The ligation reaction compensates for the weakness of reduced binding affinity resulting from splitting the aptamer, as evidenced by an approximately 2-fold lower dissociation constant than the non-ligated system. This newly developed PT-SpA-CLR strategy is further integrated with colorimetric or fluorescent reporting mechanisms to achieve easy-to-use and low-cost biosensors utilizing ligation to produce a fully active G-quadruplex or an RNA-cleaving DNAzyme to report protein binding. Both assays can achieve specific detection of an intended protein target with a limit of detection at the picomolar level even when challenged in biological samples. The combined PT-SpA-CLR and versatile sensing strategies offer attractive universal platforms for efficient detection of protein biomarkers.

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.

Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro Selection.

The Hammerhead Ribozyme (HHR) is a ubiquitous RNA enzyme that catalyzes site-specific intramolecular cleavage. While mutations to its catalytic core have traditionally been viewed as detrimental to its activity, several discoveries of naturally occurring variants of the full-length ribozyme challenge this notion, suggesting a deeper understanding of HHR evolution and functionality. By systematically introducing mutations at key nucleotide positions within the catalytic core, we generated single-, double-, and triple-mutation libraries to explore the sequence requirements and evolution of a full-length HHR. In vitro selection revealed many novel hammerhead variants, some of which possess mutations at nucleotides previously considered to be essential. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids.

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.

Higher Affinity Enables More Accurate Detection of SARS-CoV-2 in Human Saliva Using Aptamer-based Litmus Test.

Many aptamers have been generated by SELEX to recognize spike proteins of SARS-CoV-2, some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000-fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (Kd = ~10 pM) can identify the SARS-CoV-2 infection much more accurately than the dimeric aptamer assay (Kd = ~100 pM) and monomeric aptamer assay (Kd = ~10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80-100% sensitivity and 100% specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics.

Fighting Mutations with Mutations: Evolutionarily Adapting a DNA Aptamer for High-Affinity Recognition of Mutated Spike Proteins of SARS-CoV-2.

An on-going challenge with COVID-19, which has huge implications for future pandemics, is the rapid emergence of viral variants that makes diagnostic tools less accurate, calling for rapid identification of recognition elements for detecting new variants caused by mutations. We hypothesize that we can fight mutations of the viruses with mutations of existing recognition elements. We demonstrate this concept via rapidly evolving an existing DNA aptamer originally selected for the spike protein (S-protein) of wildtype SARS-CoV-2 to enhance the interaction with the same protein of the Omicron variants. The new aptamer, MBA5SA1, has acquired 22 mutations within its 40-nucleotide core sequence and improved its binding affinity for the S-proteins of diverse Omicron subvariants by > 100-fold compared to its parental aptamer (improved from nanomolar to picomolar affinity). Deep sequencing analysis reveals dynamic competitions among several MBA5SA1 variants in response to increasing selection pressure imposed during in vitro selection, with MBA5SA1 being the final winner of the competition. Additionally, MBA5SA1 was implemented into an enzyme-linked aptamer binding assay (ELABA), which was applied for detecting Omicron variants in the saliva of infected patients. The assay produced a sensitivity of 86.5% and a specificity of 100%, which was established with 83 clinical samples.

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 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.

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.

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.