Overview of context-dependent ribozyme cleavage and RT-qPCR measurements. (A) Context-dependent ribozyme (Rz) cleavage. (Top) Different genetic contexts, i.e., upstream (US) or downstream (DS) sequences, can inhibit ribozyme cleavage. Orange scissors indicate the intended ribozyme cleavage site. (Bottom) Ribozyme activity can vary in different environments, such as in vitro (IVT) and in cells. (B) Schematic of cotranscriptionally encoded RNA strand displacement (ctRSD) gates. Domains Y, j, X, and domains i′, X′, j′ represent different upstream and downstream sequences, respectively. (C) Denaturing gel electrophoresis results of ctRSD gate sequences selected to validate the RT-qPCR method. G1–G6 have different sequences upstream and/or downstream of a minimal version of the antigenomic HDV ribozyme (Schürer et al. 2002), termed Ro. See Supplemental Section 1 for gate schematics and sequences. The gel was prestained with SYBR Gold. (D) Schematic of primer layout to measure ribozyme cleavage with RT-qPCR. The PCRu primers (pink) span the cleavage site (orange dashed line) and should only amplify uncleaved products. The PCRo primers amplify both cleaved and uncleaved products to amplify total RNA. U is a reference RNA that contains a single base mutation (maroon X) compared to G, which abolishes ribozyme activity. The difference in PCRu Ct between G and U transcripts, corrected for total RNA differences by PCRo, yields a measure of ribozyme cleavage (Materials and Methods). BO represents a blocking oligonucleotide (BO) that is added to prevent ribozyme cleavage during sample preparation (Materials and Methods). The red circle at the left end of BO indicates a 3′ amino modification to prevent extension during RT-qPCR. The numbers below the box indicate the length of each domain in bases.
