A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids

Abstract

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop‐initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one‐stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost‐effective detection of low‐abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS‐CoV‐2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point‐of‐care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

Advances in rapid point-of-care virus testing

“…With a detection limit in the range of fM-aM, SPOT has demonstrated powerful performance in infection diagnostic assays. 45 Based on the integration of an isothermal cascade signal amplification strategy and a SERS-activated silver nanorods (AgNRs) sensing chip, Zhang et al proposed a one-pot SERS assay for the rapid and accurate detection of RNA from SARS-CoV-2 viruses, which achieves an ultra-high sensitivity of as low as 5.18 × 10 copies per mL in 60 min and has excellent specificity. This technology is expected to play an important role in the diagnosis of new coronaviruses, as it is not only able to recognize SARS-CoV-2 RNA from genetic mutations, but also incompatible with coronaviruses such as severe acute respiratory syndrome (SARS-CoV), Middle East Respiratory Syndrome (MERS-CoV), and other respiratory viruses (Fig.…”

Splittable systems in biomedical applications

“…99,100 Similar to split aptamers, the applications of split DNAzymes are focused on developing biosensors for protein detection and RNA detection. 101–103…”

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing

“…The accurate and timely detection of disease biomarkers is essential to ensure the effective treatment and epidemiological surveillance. − Nucleic acid testing (NAT) capable of detecting trace amount of disease-related DNA or RNA via nucleic acid amplification has become the gold standard for various infectious and somatic diseases. ,− However, standard NATs relying on polymerase chain reaction (PCR) require costly equipment and trained personnel and thus can only be used in centralized facilities . The ongoing need to enable accurate testing at home and at the point-of-care (POC) demands the field-deployable NATs with high assay performance but low infrastructural requirements. − Toward this goal, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are currently revolutionizing NATs by offering exceptional sensitivity, specificity, and accessibility. − A parrel effort was also made to engineer deoxyribozymes (DNAzymes) into CRISPR-like tools for gene editing and disease diagnostics because DNAzymes also possess RNA/DNA-cleaving activities but with no need for protein components. − However, existing DNAzymes do not possess sequence selectivity to nucleic acid targets. To address this challenge, Mokany et al introduced a multicomponent DNAzyme (MNAzyme) design that allowed the detection of any nucleic acid target by splitting the catalytic core of a DNAzyme into two parts, each of which was extended with a complementary domain to a given target nucleic acid .…”