DNA oligonucleotide input requirements for RNAPs to generate DragonRNA. RNAP reactions shown here were run under “Lu conditions” and analyzed on a 15% TBE-urea gel. (A) Several DNA oligonucleotides were designed to test sequence specificity, structure, and priming requirements of the input oligonucleotide. The sequences of these oligonucleotides are shown here, with the FAM-label, two 3′-end bases, and the hypothesized priming loci color-coded. Below: proposed configuration showing DNA oligonucleotide looping and base-pairing with itself between the two 3′-end bases and an internal complementary site, drawn here as intra-molecular. (B) Rpo41 in vitro reactions with DNA oligonucleotides from panel (A). (C) hmtRNAP in vitro reactions with DNA oligonucleotides from panel (A). Gel images were independently contrast-enhanced, spliced together, shown as a montage, and overlaid in PowerPoint with 50% transparency of the nucleic acid-stained image over the FAM image of the same gel; separate images are in Supplementary File S1.