Fig. 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-m⁷GRNA_gluc). (C) Absorbance and fluorescence HPLC profiles of crude dual labelling product Cy5-m⁷GRNA_gluc-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-m⁷GRNA_gluc-Biot (right) and electrophoretic mobility shift assay (EMSA) of crude and HPLC-isolated products of 3′ biotinylation (left).