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.

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.