Modified Nucleotides as Substrates of Terminal Deoxynucleotidyl Transferase

Abstract

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N⁴-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3'-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3'-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures.

Keywords: 4-bromo-2-pyridone; 4-chloro-2-pyridone; 4-thio-2’-deoxyuridine triphosphate; N4-amino-2’-deoxycytidine triphosphate; UV cross-linking; terminal deoxynucleotidyl transferase.

Scheme 1

Synthesis of 4-thio-2’-deoxyuridine triphosphate (3) and N⁴-amino-2’-deoxycytidine triphosphate (4). Reagents and conditions: (i) Ac₂O, NaOAc, 90 °C, 30 min; (ii) Lawesson’s reagent, toluene, 90 °C, 2 h; (iii) NaOCH₃, CH₃OH, rt, 30 min; (iv) POCl₃, Bu₃N, trimethyl phosphate, 0 °C, 90 min; then Bu₃N, (NHBu₃)₂H₂P₂O₇, CH₃CN, 0 °C, 15 min; (v) NH₂-NH₂, H₂O, rt, 18 h.

Scheme 2

Synthesis of 5-cyano- and 5-carboxy-dUTP. Reagents and conditions: (i) 1,1,1,3,3,3-hexamethyldisilazane (HMDS), trimethylsilyl chloride (TMSCl), 80 °C, 4h; then 1,3,5-O-triacetyl-2-deoxyribose, SnCl₄, rt, 5 h; (ii) NaOCH₃, CH₃OH, rt, 30 min; (iii) POCl₃, Bu₃N, trimethyl phosphate, 0–4 °C, 2–3 h; then Bu₃N, (NHBu₃)₂H₂P₂O₇, CH₃CN, 0 °C, 10–15 min.

Figure 1

Polyacrylamide gel electrophoresis (PAGE) analysis of primer extension reactions (PEX) with terminal deoxynucleotidyl transferase (TdT) in glutamate/Mg²⁺ buffer. Lane 1, primer labelled at the 5'-end with a radioactive isotope of phosphorus (5’-³³P-labelled primer); lanes 2–12, products of PEX using: lane 2, 2’-deoxythymidine triphosphate (dTTP); lane 3, 2’-deoxyuridine triphosphate (dUTP); lane 4, 2’-deoxycytidine triphosphate (dCTP); lane 5, 2-pyridone-2’-deoxyriboside triphosphate (dPyrTP); lane 6, dPyr^4OHTP; lane 7, dPyr^4ClTP; lane 8, dPyr^4BrTP; lane 9, dPyr^5COOHTP; lane 10, 4-thio-dUTP; lane 11, dU^5CNTP; lane 12, dU^5COOHTP; lane 13, N⁴-amino-dCTP.

Figure 2

PAGE analysis of PEX with TdT in cacodylate/Co²⁺ buffer. Lane 1, 5′-³³P-labelled primer; lanes 2–12, products of PEX using: lane 2, dTTP; lane 3, dUTP; lane 4, dCTP; lane 5, dPyrTP; lane 6, dPyr^4OHTP; lane 7, dPyr^4ClTP; lane 8, dPyr^4BrTP; lane 9, dPyr^5COOHTP; lane 10, 4-thio-dUTP; lane 11, dU^5CNTP; lane 12, dU^5COOHTP; lane 13, N⁴-amino-dCTP.

Figure 3

PAGE analysis of cross-linked complexes of 4-thio-dU-ONs and TdT. Lane 1, recombinant TdT; lane 2, molecular mass marker (kDa); lane 3, 4-thio-dU-ON:TdT UV-free control; lane 4, cross-linked complexes of 4-thio-dU-ON:TdT, dose of UV irradiation ~17.2 J/cm²; lane 5, cross-linked complexes of 4-thio-dU-ON:TdT, dose of UV irradiation ~4.6 J/cm²; lane 6, cross-linked complexes of 4-thio-dU-ON:TdT (excess of TdT), dose of UV irradiation ~17.2 J/cm²; lane 7; dU-ON:TdT control, dose of UV irradiation ~17.2 J/cm².