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Competition between CTP and 2’deoxy-2’fluoro-CTP.

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posted on 04.06.2020, 17:28 by Egor P. Tchesnokov, Ben A. Bailey-Elkin, Brian L. Mark, Matthias Götte

(A) RNA primer/template used in the RNA synthesis assays to test competition of CTP and 2’deoxy-2’fluoro-CTP is shown above the gel. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. Template G allows incorporation of CTP or 2’deoxy-2’fluoro-CTP and their competition for incorporation when both nucleotides are present in the reaction mixture. RNA synthesis was monitored with purified CCHFV L protein in the presence of [α-32P]GTP, RNA primer/template, 5 mM MgCl2 and 3.7, 11, 33 and 100 μM ATP, CTP, UTP and increasing concentrations of 2’deoxy-2’fluoro-CTP. The presence of three natural NTPs in the absence of 2’deoxy-2’fluoro-CTP allows full-length product formation up to position 14. Lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (B) Full-length-template product was quantified as a fraction of total signal in the lane, normalized to the full-template-length product fraction in the absence of 2’deoxy-2’fluoro-CTP and plotted versus log concentrations of the 2’deoxy-2’fluoro-CTP. Data were fitted to a dose response function in GraphPad (Prism 6.0) to determine the concentration of 2’deoxy-2’fluoro-CTP at which the amount of full-length-template product decreased by 50% (IC50).