Journal: Nucleic Acids Research

Article Title: Ethylenediamine derivatives efficiently react with oxidized RNA 3′ ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation

doi: 10.1093/nar/gkab867

Figure Lengend Snippet: Mono- and dual labelling of IVT RNA. ( A ) Scheme of the process: NTPs mix in the presence or absence of initiating dinucleotide (ARCA, N 3 -AG or N 3 -m 7 GpppG) 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-RNA 35 , N 3 -RNA 35 , N 3 -m 7 GRNA 276 and N 3 -m 7 GRNA gluc ). ( C ) Absorbance and fluorescence HPLC profiles of crude dual labelling product Cy5-m 7 GRNA 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 N 3 -m 7 GRNA gluc -Biot (right) and electrophoretic mobility shift assay (EMSA) of crude and HPLC-isolated products of 3′ biotinylation (left).

Article Snippet: Examples of such caps are commercially available anti-reverse cap analogue (ARCA), which is as dinucleotide cap 0 analogue (m 7,3 ′ - O GpppG) ( , ), or a trinucleotide cap 1 analogue (m 7,3′-O Gppp m2′-O ApG), which is used in the production of BioNTech/Pfizer mRNA COVID-19 vaccine ( ).

Techniques: Incubation, Isolation, Fluorescence, Electrophoresis, Electrophoretic Mobility Shift Assay