Nucleotide Analogues as Inhibitors of SARS-CoV Polymerase

Jingyue Ju; Xiaoxu Li; Shiv Kumar; Steffen Jockusch; Minchen Chien; Chuanjuan Tao; Irina Morozova; Sergey Kalachikov; Robert N. Kirchdoerfer; James J. Russo

As seen in Figs. 3a and 3b, when the primer-template complex (sequences shown at top of Fig. 3 ) and 2'-F,Me-UTP were incubated with the low fidelity 9 o N polymerase mutants, 29-31 Therminator II (T2) and Therminator IX (T9), we observed single product peaks with molecular weights of 5492 Da and 5488 Da, indicating single base extension in the polymerase reaction. Thus 2'-F,Me-UTP was able to be incorporated and block further nucleotide incorporation. In contrast, when the extension reactions were carried out with high-fidelity Thermo Sequenase DNA polymerase (TS), 32 there was no incorporation, as evidenced by a single primer peak at 5172 Da (Fig. 3c ). This supports our rationale that Thermo Sequenase, a high fidelity enzyme originally designed for accurate Sanger sequencing, will not incorporate 2'-F,Me-UTP, while a lowfidelity polymerase, such as T2 and T9, will incorporate 2'-F,Me-UTP and stop further nucleotide incorporation. When dTTP was used as a positive control with these three enzymes, incorporation continued past the first A in the template, resulting in a higher molecular weight peak.