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RNA-cleaving DNAzymes and their multicomponent nucleic acid enzymes (MNAzymes) have been successfully used to detect nucleic acids and proteins. The appropriate split of the catalytic cores of DNAzymes is critical to the formation of MNAzymes with high catalytic activities. However, for protein detection, no systematic investigation has been made on the effects of the split locations and secondary structures of MNAzymes on the catalytic activities of the cleavage reaction. We systematically studied how split locations and secondary structures affect the activity of the MNAzymes that catalyze multiple cleavage steps. We engineered the MNAzymes on the basis of the RNA-cleaving DNAzyme 10-23 as a model system. We designed 28 pairs of MNAzymes, representing 14 different split locations and two secondary structures: the three-arm and the four-arm structures. By comparing the multiple turnover numbers (kobs.m) of the 28 MNAzymes, we showed that the split location between the seventh cytosine and the eighth thymine of the catalytic core region and the four-arm structure resulted in optimum catalytic activity. Binding-induced DNA assembly of the optimized MNAzymes enabled sensitive detection of two model protein targets, demonstrating promising potential of the binding-assembled MNAzymes for protein analysis. The strategy of binding-assembled MNAzymes and systematic studies measuring multiple turnover numbers (kobs.m) provide a new approach to studying other partial (split) DNAzymes and engineering better MNAzymes for the detection of specific proteins.

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30–39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.