Testing priming sites for RNAPs to generate DragonRNA. (A) Several DNA oligonucleotides were designed to test priming sites and priming distance from 3′ end of the input DNA oligonucleotide. The sequences of these oligonucleotides are shown here, with the FAM-label, two 3′-end bases, and the hypothesized priming loci color-coded. (B) Rpo41 in vitro reactions with DNA oligonucleotides from panel (A). (C) hmtRNAP in vitro reactions with DNA oligonucleotides from panel (A). Note that gels show both FAM labeled and total nucleic acid, with the faster-migrating, total-nucleic-acid-stained bands that do not show the fluorescent label consistent with synthesis by canonical initiation on the DNA input. RNAP reactions shown in panels (B) and (C) were run under “NEPol conditions” and analyzed on a 15% TBE-urea gel. 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.