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. 2019 Oct 25;141(45):18104–18112. doi: 10.1021/jacs.9b08237

Figure 4.

Figure 4

Synthetic strategy to access RNAs containing a single, defined pyrophosphate linkage. RNA primer containing a terminal 3′-phosphate monoester is annealed to a chimeric DNA/RNA template. Primer extension with activated guanosine monomers generates a 3′–5′ pyrophosphate linkage. Digestion of the DNA residues of the template with Turbo DNase generates a ssRNA containing a terminal pyrophosphate linkage. Alternatively, T4 RNA ligase 2 can be used to ligate an RNA oligonucleotide downstream in excellent yields (>95%). The same DNase treatment can then be used to generate ssRNA containing an internal pyrophosphate linkage. Gray: RNA. Black: DNA. See the Supporting Information for detailed experimental procedures.