Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5′ rN18-N1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN18) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8–17 variants, identified from ∼75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ≥0.1 min−1, and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.

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.

We report as imple and rapid saliva-based SARS-CoV-2 antigen test that utilizes an ewly developed dimeric DNAa ptamer,d enoted as DSA1N5, that specifically recognizes the spike proteins of the wildtype virus and its Alpha and Delta variants with dissociation constants of 120, 290 and 480 pM, respectively,a nd binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1 pM and 2.3 pM, respectively.T o develop ah ighly sensitive test, DSA1N5 was immobilized onto gold electrodes to produce an electrochemical impedance sensor,which was capable of detecting 1000 viral particles per mL in 1:1d iluted saliva in under 10 min without any further sample processing. Evaluation of 36 positive and 37 negative patient saliva samples produced aclinical sensitivity of 80.5 % and specificity of 100 %a nd the sensor could detect the wildtype virus as well as the Alpha and Delta variants in the patient samples,w hich is the first reported rapid test that can detect any emerging variant of SARS-CoV-2.

We report a signal amplification strategy termed DNAzyme feedback amplification (DFA) that takes advantage of rolling-circle amplification (RCA) and an RNA-cleaving DNAzyme (RCD). DFA employs two specially programmed DNA complexes, one composed of a primer and a circular template containing the antisense sequence of an RCD, and the other composed of the same circular template and an RNA-containing substrate for the RCD. RCA is initiated at the first complex to produce RCD elements that go on to cleave the substrate in the second complex. This cleavage event triggers the production of more input complexes for RCA. This reaction circuit continues autonomously, resulting in exponential DNA amplification. We demonstrate the versatility of this approach for biosensing through the design of DFA systems capable of detecting a microRNA sequence and a bacterium, with sensitivity improvements of 3-6 orders of magnitude over conventional methods.

The reliable detection of pathogenic bacteria in complex biological samples using simple assays or devices remains a major challenge. Herein, we report a simple colorimetric paper device capable of providing specific and sensitive detection of Helicobacter pylori (H. pylori), a pathogen strongly linked to gastric carcinoma, gastric ulcers, and duodenal ulcers, in stool samples. The sensor molecule, an RNA‐cleaving DNAzyme obtained through in vitro selection, is activated by a protein biomarker from H. pylori. The colorimetric paper sensor, designed on the basis of the RNA‐cleaving property of the DNAzyme, is capable of sensitive detection of H. pylori in human stool samples with minimal sample processing and provides results in minutes. It remains fully functional under storage at ambient temperature for at least 130 days. This work lays a foundation for developing DNAzyme‐enabled paper‐based point‐of‐care diagnostic devices for monitoring pathogens in complex samples.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.

Summary To avoid false alarms for vibration‐based structural damage detection methods, temperature effects on damage‐sensitive features should be eliminated. In this paper, a novel two‐step damage identification method combining a multilayer neural network and novelty detection is developed to differentiate the changes in natural frequencies (one of the most commonly used damage features that can be obtained reliably and relatively easily) due to damage from those induced by temperature variations. In the first step, a multilayer artificial neural network, which resembles an auto‐associative neural network but uses temperature variables in addition to the frequencies as the inputs, is explored to identify patterns in frequencies of undamaged structures under varying temperatures. Euclidean distance is then utilized as a novelty index to quantify the discordancy between patterns in undamaged cases and candidate cases. Numerical studies using a simply supported beam and finite element models based on an experimental grid structure, which simulate different levels of stiffness reductions under varying temperature conditions, are used to verify the detectability and robustness of the proposed approach. It is shown that the incorporation of the proposed artificial neural network with novelty detection enables one to robustly distinguish damage occurrence and severity regardless of temperature variations and noise perturbations. Using an unsupervised learning scheme, the proposed approach transforms a multivariate analysis using modal frequencies and temperature data into a straightforward univariate discordancy test using the novelty index. Given these competitive advantages, this approach is very attractive for the development of an automated continuous monitoring system in practical applications.

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml−1, thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures.