Typhoon 9500 imaging system

In this assay, 50 μl of the total reaction volume containing 25 nM of renatured FAM-labelled MeORNA–HNA aptamers or control solutions without or with 100 nM of rVEGF₁₆₄ in SB was incubated for 2 h at RT upon shaking at 300 rpm. After incubation, 10 μl of 6× purple gel loading dye was added to the samples and 12 μl of the final solutions was run on a 6% native polyacrylamide gel with 0.5× TBE (running buffer) at 150 V and ∼18°C until the purple tracking dye reached of the gel (∼ 3.5 h). The gel was scanned using the Typhoon 9500 imaging system (Cy2-channel) and quantified with the ImageQuant TL v8.1 software (both from GE Healthcare Life Science).

Either 100 pM of 87-mer ssDNA (Template-1) or 0.2 ng of pET-3a plasmid (Template-2) was PCR amplified in 20 μl of 1 × ThermoPol buffer with 0.2 μM of the corresponding primers (Supplementary Table S2), 200 μM of either natural dNTPs (positive control; PC) or different combinations of modified dZTPs, and 25 U/ml of Taq DNA polymerase. If specified, different additives (0.3 ng of pET-3a plasmid, 2 mM Mg²⁺, 2% DMSO, and 10% GC enhancer) were used for the PCR amplification of Template-2. The samples were first denatured at 95°C for 60 s, followed by 30 cycles of repetitive denaturation at 95°C for 30 s, annealing at 54°C (50°C for Template-2) for 15 s, extension at 68°C for 15 s, and final extension at 68°C for 10 min. The final PCR products were quenched with a loading buffer (90% formamide, 50 mM EDTA, 0.05% bromophenol blue) and analyzed by 15% denaturing PAGE. The gels of the PCR products obtained using Template-1 were scanned using a Typhoon 9500 imaging system under Cy2 and Cy3 channels and further analyzed by the ImageQuant TL v8.1 Software (both from GE Healthcare Life Science). The relative PCR amplification efficiencies were estimated using the positive control (unmodified PCR product) as 100%. The gels of the PCR products using Template-2 were first stained with 1 × SYBR Gold Nucleic Acid Gel Stain and then analyzed using the above-described method.