Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA‐based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA‐cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation‐inspired molecular device for controlling gene expression and cancer treatment.

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Therapeutic nucleic acid agents (TNA) can be activated by a marker RNA sequence followed by initiation of targeted RNA cleavage. This property can be used in conditional cell suppression, e. g., cancer marker‐dependent cell death. However, healthy cells often express lower levels of cancer markers, thus jeopardizing TNA activation exclusively in cancer cells. Earlier, we developed a conditionally activated split deoxyribozyme construct (DNA thresholder or DTh) that can be activated by high but not by low concentrations of cancer markers. It's activity, however, was suppressed by very high marker concentrations. Herein, we combine the DTh functional units in a single DNA association (Thresholding DNA nanomachine or Th‐DNM). Th‐DNM maintains a high level of RNA cleavage activity in the presence of marker concentrations above the threshold level. Th‐DNM differentiated fully complementary miR17 markers sequence from double base mismatched miR‐20. Th‐DNM can become a building block of DNA nanorobots for cancer treatment.

Nucleic acid‐based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein‐enzyme‐free nano‐construction, named four‐armed DNA machine (4DNM), that requires neither an amplification stage nor a high‐temperature annealing step for SARS‐CoV‐2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA‐binding arms. Additional arms were found to improve the limit of detection at least 10‐fold. The sensor distinguished SARS‐CoV‐2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT‐qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point‐of‐care or home diagnostic test.