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. 2021 Sep 30;50(1):e3. doi: 10.1093/nar/gkab867

Figure 3.

Figure 3.

Mono- and dual labelling of IVT RNA. (A) Scheme of the process: NTPs mix in the presence or absence of initiating dinucleotide (ARCA, N3-AG or N3-m7GpppG) are incubated with gene-coding DNA template in presence of T7 RNA polymerase. After isolation from IVT mixture, RNA products are subjected to 3′ labelling (top) or simultaneous 5′+3′ dual labelling (bottom). (B) Representative HPLC chromatograms of crude labelling products of Cy3 3′ labelling (top) or Cy5/Cy3 5′+3′ dual labelling (bottom) performed on 35, 276 and 993 nucleotide-long RNAs (ppp-RNA35, N3-RNA35, N3-m7GRNA276 and N3-m7GRNAgluc). (C) Absorbance and fluorescence HPLC profiles of crude dual labelling product Cy5-m7GRNAgluc-Cy3 with designated retention times of collected fractions (right) and agarose electrophoresis of concentrated HPLC fractions (left); (D) HPLC chromatogram of crude 3′-biotinylated mRNA N3-m7GRNAgluc-Biot (right) and electrophoretic mobility shift assay (EMSA) of crude and HPLC-isolated products of 3′ biotinylation (left).