Improved detection of ac4C in total RNA through noncanonical dNTPs. (A) Predicted base-pairing interactions involving the noncanonical NTPs 2-amino-dATP (2-NH2), 2-hydroxy-dATP (2-OH), or 2-amino-purineTP (2-AP). (B) RT-PCR of HeLa cell total RNA with primers flanking a fully acetylated ac4C site in 18S rRNA helix 45. PCR was performed with the canonical dNTPs or with dGTP either partially (75% analog:25% dGTP) or fully substituted by the indicated analog. (C) Reduction of an RNA oligo containing a single C or ac4C and cDNA synthesis with HIV RTase and the canonical dNTPs, or with dGTP either partially (75%:25%) or fully substituted by the indicated analog. Primer extension assay with a radiolabeled primer and polyacrylamide gel electrophoresis. (D) Quantification of the primer extension results from NaCNBH3-treated RNA in C comparing the signal intensity associated with unincorporated primer or full-length cDNA as a percent of total lane volume. Volumes were derived from identically sized boxes in ImageQuant. (E) NaBH4 and NaCNBH3 reduction of total RNA and cDNA synthesis with TGIRT, SSIV, or HIV RTase with the canonical dNTPs (100% dGTP) or with dGTP partially substituted by the indicated analog (75% analog:25% dGTP). PCR amplification with primers flanking a ∼100% acetylated site in 18S rRNA helix 45 and Sanger sequencing results surrounding the known ac4C sites. (F) Quantification of C:T mismatch levels through chromatogram peak heights at 18S rRNA nt 1842 from E.
