Supplementary method accompany nupack codes.docx

we introduce an advanced conditional platform named intelligent guide RNA (IngRNA) that exhibits the capacity to sense, make decisions, and execute intelligent actions. In this platform, the CRISPR RNA (CrRNA) sequence is sequestered by flanking sequences to prevent its function from targeting genes of interest in the absence of the trigger RNA. The design includes a short linker sequence, with the trigger RNA site located upstream. A substantial portion of these specific sequences are intentionally inhibited by the complementary strand. The upper loop of the platform contains the first toe-hold, which provides the necessary binding force for the trigger RNA. The trigger RNA binding event initiates the opening of the second toe-hold, situated within the complementary strand of the trigger RNA binding site. The liberation of the second toe-hold from its obstructed state facilitates the connection of the 5' side of the platform to it, resulting in the release of CrRNA from its restrained condition, thus enabling it to carry out its intended function. In this biological circuit, the targeting of the luciferase gene is investigated in the presence or absence of cognate trigger RNA in test tubes, bacteria, and mammalian cells.