Exonuclease 8

The l-DNA oligonucleotides were purchased from ChemGenes or synthesized by a Mermade 192E DNA/RNA synthesizer (BioAutomation, Irving, TX, USA). The PCR reactions were performed in 20 μl reaction systems containing 50 mm HEPES (pH 7.5), 5 mm MgCl₂, 50 mm NaCl, 0.1 mm EDTA, 5 mm DTT, 10% glycerol, 3% DMSO, 0.1 mg ml⁻¹ BSA, 100 μm (each) L-dNTPs, 0.5 μm (each) l-primers, 20 nml-ssDNA template and ~500 nmd-Dpo4-5m polymerase. Because the mirror-image DNA polymerization was apparently less efficient than the corresponding natural system, likely due to the lower purity of l-dNTPs which was also observed in previous studies on the ASFV pol X system [1 (link)], an extension time of 1 h was used. The PCR program settings were 86 °C for 3 min (initial denaturation); 86 °C for 30 s (denaturation), 58 °C for 1 min (annealing) and 65 °C for 1 h (extension) for 40 cycles. The products were analyzed by 3% sieving agarose gel electrophoresis and stained by GoldView (Solarbio, China). Both the products of miPCR and PCR (5 μl) were digested by 0.5 U DNase I (NEB, Ipswich, MA, USA) at 37 °C for 10 min, or Exonuclease VIII, truncated (NEB) at 37 °C for 4 h, and analyzed by 3% sieving agarose gel electrophoresis.

The TN-RCA reaction product was digested with MseI (NEB) (10 U) for 20 min at 37 °C in the presence or absence of cutting primer, Msecutprimer: 5’-TTTATCTTAACTCACCAACT-3’ (underlined: MseI recognition site), and the enzyme subsequently inactivated at 65 °C for 20 min. Lambda exonuclease (NEB) (0.5 U), exonuclease III (NEB) (20 U), exonuclease VIII (NEB) (2 U), T7 exonuclease (2 U), RNase H (NEB) (1 U), RNase A (Thermo Scientific, Waltham, MA, USA) (10 U), RNase A/T1 Mix (Thermo Scientific) (1 U), RNase T1 (Invitrogen/Ambion) (1 U), ShortCut RNase III (NEB) (0.4 U) were added to the TN-RCA reaction mixture after the ligation step was completed, or at various times during the TN-RCA reaction. In some experiments, ShortCut RNase III (NEB) (0.4 U) was also added before the ligation step to fragment the target genomic Zika RNA. Random hexamers (Exo-Resistant Random Primer; 0.2 μL of 500 μM stock; Thermo Scientific) also was added after the ligation step.