Examining the NTA activity and nucleotide preference of MarathonRT. (A) Outline of the experimental design for the NTA assay. A DNA/RNA blunt end duplex serves as the substrate, and the arrow of the DNA strand denotes the direction of NTA. The potential NTA products are described as different numbers of nucleotide additions as shown in the figure. (B) A representative gel figure showing the separation of NTA products of 1, 2, and 3 nucleotide additions (+1, +2, and +3) from the DNA primer (p) on a high-resolution polyacrylamide gel. An equimolar dNTP mix was used in this reaction. (C), (D), (E), and (F) Representative gel figures showing the separated NTA products from the DNA primer when using individual dATP, dGTP, dCTP, and dTTP in the reaction, respectively. (G) Regression modeling the production of one, two, and three adenosine additions to calculate the rate constant for each NTA product. (H) Regression modeling the production of one and two guanosine additions to calculate the rate constant for each of them. The curves were generated from the averaged result of three repeats for each reaction.
