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.
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