Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

Abstract

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Scheme 1

Synthesis of C5-modified dUTP analogs: (i) H₂N-(CH₂)_n-NH₂ (n = 2, 4, 6), methanol, 50°C, 2 h overnight, followed by ethyl trifluoroacetate, triethylamine, room temperature (rt), 1.5–3 h; (ii) sodium methoxide, methanol, rt, 1.5 h; (iii) POCl₃, N,N,N′,N′-tetramethyl-1, 8-naphthalendiamine, trimethyl phosphate, 0°C, 45 min, followed by n-tributylamine pyrophosphate, dimethylformamide, rt, 1 h; (iv) S-ethylthiourea, triethylamine, dimethylformamide, rt, 4 h.

Scheme 2

Synthesis of C5-modified analogs of dUTP and dCTP: (i) 7 M aqueous ammonia, rt, 1 h; (ii) POCl₃, N,N,N′,N′-tetramethyl-1, 8-naphthalendiamine, trimethyl phosphate, 0°C, 45 min, followed by n-tributylamine pyrophosphate, dimethylformamide, rt, 1 h; (iii) POCl₃, dry pyridine, rt, 4 h, then concentrated aqueous ammonia, 50°C, 2 h; (iv) 1 N aqueous KOH, 86°C, 1 h; chlorotrimethylsilane, dry methanol, 31°C, 1.5 h; (v) H₂N-(CH₂)_n-NH₂ (n = 2, 4, 6), methanol, 50°C, 2 h overnight, followed by ethyl trifluoroacetate, triethylamine, rt, 1.5–3 h; (vi) S-ethylthiourea, triethylamine, dimethylformamide, rt, 4 h.

Figure 1

Sequences of amplifying regions of the pUC18 template DNA. Primer sequences are underlined.

Figure 2

Sequences of primers and templates used for standing-start experiments. ‘t’ indicates 5-(2-(6-aminohexylamino)-2-oxoethyl)-dU.

Figure 3

Representative ultraviolet images from ethidium bromide-stained PAGE gels of the 110 nt PCR product derived from pUC18 (amplifying region I) and the C5-modified nucleoside triphosphates. (A–C) The reaction mixtures containing dATP, dGTP, dCTP and T^AL (lane 3), T^AC (lane 4), T^AF (lane 5), T^PA (lane 6), T^PN (lane 7), T^PR (lane 8), T^A2 (lane 11), T^A4 (lane 12), T^A6 (lane 13), T^G6 (lane 14), T^ME (lane 15), T^CN (lane 16), or T^DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures do not contain natural TTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and dCTP; lanes 1 and 9). (D–F) Reaction mixtures containing dATP, dGTP, TTP and C^AL (lane 3), C^AC (lane 4), C^AF (lane 5), C^PA (lane 6), C^PN (lane 7), C^PR (lane 8), C^A2 (lane 11), C^A4 (lane 12), C^A6 (lane 13), C^G6 (lane 14), C^ME (lane 15), C^CN (lane 16) or C^DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures did not contain natural dCTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and TTP; lanes 1 and 9). The thermostable DNA polymerases used were Taq (A, D), Vent(exo-) (B and E), and KOD Dash (C and F).

Figure 4

Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with (A) Taq DNA polymerase, (B) Tth DNA polymerase, (C) Vent(exo-) DNA polymerase, (D) KOD Dash DNA polymerase and (E) KOD(exo-) DNA polymerase. The x-axis indicates the kind of triphosphate analog used, and the y-axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.

Figure 5

Decrease in the apparent relative catalytic efficiencies (k_cat/K_m)_rel according to the numbers of modified dUs (t) on the primers and templates. Values were obtained from standing-start experiments using the following: P0–2, TA, T^A6 and Vent(exo-) DNA polymerase (filled circles); PA, T0–3, dATP and Vent(exo-) DNA polymerase (open circles); P0–2, TA, T^A6 and KOD(exo-) DNA polymerase (filled triangles); or PA, T0–3, dATP, and KOD(exo-) DNA polymerase (open triangles).