RNA polymerization actuating nucleic acid membrane (RANAM)-based biosensing for universal RNA virus detection.

The coronavirus disease (COVID-19) pandemic has shown the importance of early disease diagnosis in preventing further infection and mortality. Despite major advances in the development of highly precise and rapid detection approaches, the time-consuming process of designing a virus-specific diagnostic kit has been a limiting factor in the early management of the pandemic. Here, we propose an RNA polymerase activity-sensing strategy utilizing an RNA polymerization actuating nucleic acid membrane (RANAM) partially metallized with gold for colorimetric RNA virus detection. Following RANAM-templated amplification of newly synthesized RNA, the presence of the RNA polymerase was determined by visualization of the inhibition of an oxidation/reduction (redox) reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and blocked Au3+. As a proof of concept, a viral RNA-dependent RNA polymerase (RdRP), which is found in various RNA virus-infected cells, was chosen as a target molecule. With this novel RANAM biosensor, as little as 10 min of RdRP incubation could significantly reduce the colorimetric signal. Further development into an easy-to-use prototype kit in viral infection diagnosis detected RdRP present at levels even as low as 100 aM. Color formation based on the presence of RdRP could be simply and clearly confirmed through smartphone-assisted color imaging of the prototype kit. This study provides a non-PCR-based RNA virus detection including its variants using RdRP-mediated polymerization.

Rapid Diagnosis of Coronavirus by RNA-Directed RNA Transcription Using an Engineered RNA-based Platform.

A coronavirus disease (COVID-19) outbreak associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading widely through person-to-person transmission. Various detection approaches have been developed involving quantitative polymerase chain reaction (qPCR) methods, CRISPR-based systems, and direct targeting of specific coronavirus proteins. However, there have only been a few reports on the detection of RNA-dependent RNA polymerase (RdRP), the primer-independent RNA-replicable protein produced by the RNA genes of coronavirus. Here, we introduce a novel diagnostic methodology for COVID-19 using the RNA-directed and de novo RNA replicable function of RdRP. We devised an RNA platform for RdRP-induced transcription (RPRIT) that includes an RNA template that can be directly transcribed by RdRP. By utilizing RPRIT, the presence of RdRP can be readily confirmed within 30 min using isothermal incubation without PCR. This RdRP detection method can provide a new route for rapid diagnosis of RNA virus-infected patients.

Double Controlled Release of Therapeutic RNA Modules through Injectable DNA-RNA Hybrid Hydrogel.

Advances in the DNA nanotechnology have enabled the fabrication of DNA-based hydrogels with precisely controlled structures and tunable mechanical and biological properties. Compared to DNA hydrogel, preparation of RNA-based hydrogel remains challenging due to the inherent instability of naked RNA. To overcome these limitations, we fabricated a DNA-RNA hybrid hydrogel via stepwise dual enzymatic polymerization. Multimeric short hairpin RNAs (shRNAs) were hybridized with functional DNA aptamers for targeting and mechanical properties of the hydrogel. The obtained DNA-RNA hybrid hydrogel was ultrasoft, robust, and injectable hence reconfigurable into any confined structures. As a model system, the hydrogel was able to mimic microtubule structures under physiological conditions and designed to release the functional small interfering RNA (siRNA)-aptamer complex (SAC) sequentially. In addition, we encoded restriction enzyme-responsive sites in DNA-RNA hybrid hydrogel to boost the release of SAC. This novel strategy provides an excellent platform for systematic RNA delivery through double-controlled release, SAC release from hydrogel, and subsequent release of siRNA from the SAC, which has promising potential in RNA therapy.