Synthesis of 2-Substituted Adenosine Triphosphate Derivatives and their use in Enzymatic Synthesis and Postsynthetic Labelling of RNA

Abstract

A series of adenosine triphosphate (ATP) derivatives bearing chloro, fluoro, amino, methyl, vinyl, and ethynyl groups at position 2 are synthesized and tested as substrates for RNA and DNA polymerases. The modified nucleotides work well in in vitro transcription with T7 RNA polymerase and primer extension (PEX) using engineered DNA polymerases (TGK, 2M) except for the bulkier 2-vinyl- and 2-ethynyl-ATP derivatives that give truncated products. However, in single nucleotide incorporation followed by PEX, they still can be used for site-specific incorporation of reactive modifications into RNA that can be further used for postsynthetic labeling through thiol-ene or Cu-catalyzed alkyne-azide cycloadditions reactions. All modified ATPs work in polyadenylation catalyzed by poly(A) polymerase to form long 3'-polyA tails containing the modifications that also can be used for labeling.

Keywords: DNA polymerases; RNA polymerases; click reactions; nucleosides triphosphates; nucleotides; polyA polymerase; thiol‐ene addition.

Scheme 1

Synthesis of modified r ^R NTPs through A) triphosphorylation of commercial nucleosides or B) through cross‐coupling reactions at position 2. Reagents and conditions: i) 1. POCl₃, PO(OMe)₃, 0 °C, 3 h; 2. (Bu₃NH)₂H₂P₂O₇, Bu₃N, DMF, 0 °C, 1 h; 3. 1 m TEAB, 0–22 °C, 1 min; ii) potassium vinyl(trifluoro)borate, Cs₂CO₃, TPPTS, Pd(OAc)₂, H₂O/MeCN (2:1), 80 °C, 2.5 h; iii) 1. TMS‐acetylene, PdCl₂(PPh₃)₂, CuI, Et₃N, DMF, rt, 3 h; 2. K₂CO₃, MeOH, 22 °C, 1.5 h; iv) DMAP, TEA, Ac₂O, MeCN, 22 °C, 1 h; v) Me₄Sn, Pd(PPh₃)₄, NMP, 80 °C, 2 h; vi) K₂CO₃, MeOH, 22 °C, overnight.

Figure 1

A) General scheme of IVT syntheses of modified RNA. B) 15% dPAGE of 70RNA_ ^R A products of IVT. Lane 1, L ‐ ladder; Lane 2, +—positive control (four natural rNTPs); Lane 3, A‐—negative control (rGTP, rCTP, rUTP); Lane 4, ^Cl A–IVT with ^Cl ATP, rGTP, rCTP, rUTP; Lane 5, ^NH2 A–IVT with r ^NH2 ATP, rGTP, rCTP, rUTP; Lane 6, ^F A–IVT with r ^F ATP, rGTP, rCTP, rUTP); Lane 7, ^V A–IVT with r ^V ATP, rGTP, rCTP, rUTP; Lane 8, ^E A–IVT with r ^E ATP, rGTP, rCTP, rUTP; Lane 9, ^Me A–IVT with r ^Me ATP, rGTP, rCTP, rUTP. SYBR Gold staining.

Figure 2

A) General scheme of PEX experiments and following RNAs generation. B) 15% dPAGE of 5′‐(6‐FAM)‐labeled 31NA_ ^R A after PEX reaction with TGK polymerase at 65 °C. Lane 1, P ‐ primer; Lane 2, +—positive control—PEX with four natural rNTPs; Lane 3, A‐—negative control—PEX with rGTP, rCTP, rUTP; Lane 4, ^Cl A–PEX with r ^Cl ATP, rGTP, rCTP, rUTP; Lane 5, ^NH2 A–PEX with r ^NH2 ATP, rGTP, rCTP, rUTP; Lane 6, ^F A–PEX with r ^F ATP, rGTP, rCTP, rUTP; Lane 7, ^V A–PEX with r ^V ATP, rGTP, rCTP, rUTP; Lane 8, ^E A–PEX with r ^E ATP, rGTP, rCTP, rUTP; and Lane 9, ^Me A–PEX with r ^Me ATP, rGTP, rCTP, rUTP.

Figure 3

A) General scheme of SNI experiments with following RNAs generation. B) 15% dPAGE of 5′‐(6‐FAM)‐labeled 16NA_ ^R A and 31NA_ ^R A after SNI and PEX reactions with 2M polymerase. Lane 1, P ‐ primer; Lane 2, +—positive control—SNI with natural rATP; Lane 3, A‐—negative control—water instead of any rNTP; Lane 4, ^V A–SNI with r ^V ATP; Lane 5, ^E A–SNI with r ^E ATP; and Lanes 6–9, PEX reactions of SNI products (from Lanes 2–5) with addition of rGTP, rCTP, and rUTP.

Figure 4

A) Schematic representation of postsynthetic modification reaction with Cy3–N₃. B) 15% dPAGE analysis of SNI PEX product 31RNA_SNI_ ^E A and the product of subsequent CuAAC reaction 31RNA_SNI_ ^Cy3 A (FAM and Cy3 scan), conversion 69%. C) Normalized emission spectra of 31RNA_SNI_ ^Cy3 A compared to 31RNA_SNI_ ^E A before postsynthetic modification and after negative control reaction of nonmodified RNA with Cy3–N₃. D) Schematic representation of postsynthetic modification reaction with CM–SH. E) 20% dPAGE analysis of SNI PEX product 31RNA_SNI_ ^V A and the product of subsequent thiol‐ene reaction 31RNA_SNI_ ^CM A, conversion 89%. Uncropped gels are given in Supporting Information, Part 3.

Figure 5

A) General scheme of polyadenylation experiments. B) 10% dPAGE of RNA products after polyadenylation reaction. Lanes 1,2, L ‐ ladder; Lane 3, +—positive control (natural rATP); Lane 4, A‐—negative control (water instead of natural or modified rATP); Lane 5, ^Cl A–r ^Cl ATP incorporation; Lane 6, ^NH2 A–r ^NH2 ATP incorporation; Lane 7, ^F A–r ^F ATP incorporation; Lane 8, ^V A–r ^V ATP incorporation; Lane 9, ^E A–r ^E ATP incorporation; and Lane 10, ^Me A–r ^Me ATP incorporation. SYBR Gold staining.

Figure 6

A) Schematic representation of postsynthetic modification reaction with Cy3–N₃. B) 15% dPAGE analysis of polyadenylation reaction product 35RNA_polyA ^E A and the product of subsequent CuAAC reaction 35RNA_polyA ^Cy3 A (Cy3 scan before and after SYBR Gold staining). C) Normalized emission spectra of 35RNA_polyA ^Cy3 A compared to 35RNA_polyA ^E A before postsynthetic modification and nonmodified 35RNA_polyA after negative control reaction with Cy3–N ₃ . D) Schematic representation of postsynthetic modification reaction with CM–SH. E) 15% dPAGE analysis of polyadenylation reaction product 35RNA_polyA ^V A and the product of subsequent thiol‐ene reaction 35RNA_polyA ^CM A (Cy3 scan after SYBR Gold staining). F) Normalized emission spectra of 35RNA_polyA ^CM A compared to 35RNA_polyA ^V A before postsynthetic modification and nonmodified 35RNA_polyA after negative control reaction with CM–SH. Uncropped gels can be seen in Supporting Information, Part 3.