De novo 3D models of SARS-CoV-2 RNA elements and small-molecule-binding RNAs to guide drug discovery

Ramya Rangan; Andrew M. Watkins; Wipapat Kladwang; Rhiju Das

For cDNA synthesis, 2.5 μL resuspension of purified, polyA magnetic beads carrying chemically modified RNA was mixed with 2.5 μL of reverse transcription premix with SuperScript-III (Thermo Fisher). The reaction was incubated at 48 °C for 45 minutes. The RNA was then degraded by adding 5 μL of 0.4 M NaOH and incubating the mixture at 90 ℃ for 3 minutes. The degradation reaction was placed on ice and quickly quenched by the addition of 2 μL of an acid quench solution (1.4 M NaCl, 0.6 M HCl, and 1.3 M NaOAc). Bead-bound, FAM labeled cDNA was purified by magnetic bead separation, washed twice with 100 μL of 70% EtOH, and air-dried for 10 minutes. To elute the bound cDNA, the magnetic beads were resuspended in 10.0625 μL ROX/Hi-Di (0.0625 μL of ROX 350 ladder [Applied Biosystems] in 10 μL of Hi-Di formamide [Applied Biosystems]) and incubated at room temperature for 20 minutes. The resulting eluate was loaded onto capillary electrophoresis sequencers (ABI-3100 or ABI-3730) either on a local machine or through capillary electrophoresis (CE) services rendered by ELIM Biopharmaceuticals.