Fig. S1.
Incorporation of DMSO and 2´ O-methyl–modified templates eliminates self-templated run-on transcription by T7 RNAP. (A) Secondary structure of the 5′-L344-UUG RNA construct (lr-AID mutations are shown in red) including the proposed self-coded run-off transcription product (blue). One-dimensional proton spectra of U5:AUG control oligo-RNAs containing either the UUA (B) or UUG (C) lrAID motif. (D) One-dimensional proton spectrum of 5′-L344-UUG RNA prepared without addition of DMSO or a 2´ O-methyl–modified template. The signal at 6.48 ppm (highlighted with yellow box) is inconsistent with the expected UUG-UAA base pairing engineered at U5:AUG but rather is consistent with UUA–UAA base pairing that would result from self-coded 3′ extension of the RNA. (E) Run-off transcription is prevented by the inclusion of DMSO and 2´ O-methyl–modified template in the transcription reaction. The absence of a signal at 6.48 ppm in the 1D proton spectrum of the 5′-L344-UUG RNA prepared by this method is consistent with the elimination of run-off transcription at the 3′ terminus of the RNA.