In vitro Synthesis of Nucleic Acid Derivatives

All chemicals were either purchased from Merck or Jena Biosciences and used without further purification. Oligonucleotides were purchased from Generi Biotech. In vitro transcription was performed using a modification of a previously published method,^{9 (link)} in a 25 μL mixture containing: 80 ng μL⁻¹ of template DNA (35A for Ap_2–5A, Gp_3–4A, NAD(H), CoA, ADP-ribose and m⁷Gp₃A, or 35G for Gp_3–4G, Ap_3–5G, and m⁷Gp₃G), 1 mM UTP, 1 mM CTP, 1 mM ATP, 0.8 mM GTP and 0.5 μL α ³²P GTP (activity: 9.25 MBq in 25 μL), 1.6 mM of Np_nNs (Ap_2–5A, Gp_3–4G, Ap_3–5G, m⁷Gp₃G, m⁷Gp₃A, ADP-ribose), or 8 mM of cofactors (3′-dpCoA, NAD and NADH), 5% DMSO, 0.12% triton X-100, 12 mM DTT, 4.8 mM magnesium chloride and 10× reaction buffer for T7 RNAP (40 mM Tris-HCl, 6 mM MgCl₂, 1 mM DTT, 2 mM spermidine, pH 7.9 at 25 °C) and 62.5 units of T7 RNAP. The mixture was incubated for 2 h at 37 °C.