Kinase experiment:

Thermal DNA duplex denaturation studies are performed with templates containing up to twelve consecutive dA residues that are paired with its complement template containing consecutive T or Pseudothymidine (ψT) residues. Experiments are performed in a buffer (45 mM NaCl, 45 mM sodium citrate, pH 8.1, final vol. 1.5 mL) containing template and its complement (1.5 μM of each). Absorbance (260 nm) is monitored over a range of 25.0 to 90.0℃ with a change in temperature of 0.5℃/min for five heating cycles. The initial heating cycle is discarded and the Tm is determined by averaging the temperatures of the remaining four cycles. The ΔTm between similar duplexes is calculated by subtracting the Tm of the duplex containing standard bases from the Tm of the duplex containing C-glycosides (including Pseudothymidine)[2].

Pseudothymidine is a C-nucleoside analog of thymidine.

Pseudothymidine is a C-nucleoside analog of thymidine[1]. The calculated δδG°50/mod is -0.5 kcal/mol, with a δTm/mod of 0.82°C. For the duplexes containing nine dA-T/ψT pairs, the δTm/mod is -0.9°C and a δδG°50/mod is +1.1 kcal/mol. The modification of the duplex containing 12 consecutive dA-T/ψT base pairs produces a δTm/mod of -0.9°C and a δδG°50/mod of +1.2 kcal/mol[2].

[1]. S Lutz, et al. An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases. Nucleic Acids Res. 1999 Jul 1; 27(13): 2792-2798. [2]. Havemann SA, et al. Incorporation of multiple sequential pseudothymidines by DNA polymerases and their impact on DNA duplex structure. Nucleosides Nucleotides Nucleic Acids. 2008 Mar;27(3):261-78.