Evaluation of 3'-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides

Abstract

Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis. So far, most approaches strived to improve controlled enzymatic synthesis of canonical DNA and no potential routes to access xenonucleic acids (XNAs) have been reported. In this context, we have investigated the possibility of using phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. Phosphate is ubiquitously employed in natural systems and we demonstrate that this group displays most characteristics required for controlled enzymatic synthesis. We have devised robust synthetic pathways leading to these challenging compounds and we have discovered a hitherto unknown phosphatase activity of various DNA polymerases. These findings open up directions for the design of protected DNA and XNA nucleoside triphosphates for controlled enzymatic synthesis of chemically modified nucleic acids.

Fig. 1. Blocking enzymatic synthesis with a 3′-phosphorylated primer.

Gel image (PAGE 20%) shows the result of the PEX reactions with 3′-phosphorylated primer P1 and template T1 with natural dNTPs and different DNA polymerases. Natural triphosphates were at a final concentration of 200 µM. The following quantities of polymerases and reaction conditions were used: Phusion (2 U), Hemo Klen Taq (8 reactions), Taq (5 U), Bst (8 U), Q5 (2 U), Therminator (2 U), Vent (exo⁻) (2 U): 60 °C, 15 min and 30 min; Dpo4 (2 U), Deep Vent (2 U): 55 °C, 15 min and 30 min; Kf (exo⁻) (5 U): 37 °C, 15 min and 30 min. Negative control (T−): No polymerase added to the mixtures or reactions only with dATP and dCTP or dATP, dCTP, and dTTP only. Positive control (T+): with all natural nucleotides and Taq polymerase. All reactions were incubated at adequate reaction temperatures for 1 h. P represents unreacted, 5′-FAM-labeled primer.

Fig. 2. Synthesis of 3′-phosphate-dTTP 5 and 3′-phosphate-LNA-TTP 10.

A Synthetic scheme for the synthesis of nucleotide 5 and (B) synthetic pathway leading to nucleotide 10. Reagents and conditions: (i) ETT, CH₃CN, H₂O (9:1), rt, 10 min, quantitative for both 2 and 7; (ii) I₂, pyridine, H₂O (9:1), rt, 60 min, quantitative for both 3 and 8; iii) TFA, DCM, rt, 60 min, 4 (84%), 9 (97%); iv) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; 2. (nBu₃NH)₂ H₂P₂O₇, DMF, nBu₃N, rt, 45 min; 3. I₂, pyridine, H₂O, rt, 30 min; 4. NH₄OH, MeNH₂ (1:1), rt, 2 h, 12% over 4 steps for both 5 and 10.

Fig. 3. Evaluation of modified nucleotides 5 and 10 under PEX reaction conditions.

Gel image (PAGE 20%) shows the results of PEX reactions with 3′-phos-dTTP 5 and 3’-phos-LNA-TTP 10 with 5′-FAM-labeled primer P1 (devoid of a 3′-phosphate moiety), template T1, and various DNA polymerases. Natural and modified triphosphates were at a final concentration of 200 µM. The following quantities of polymerases were used: Phusion (2 U), Hemo Klen Taq (8 reactions), Taq (5 U), Bst (8 U), Q5 (2 U), Therminator (2 U), Vent (exo⁻) (2 U): 60 °C, 1 h; Dpo4 (2 U), Deep Vent (2 U): 55 °C, 1 h; Kf (exo⁻) (5 U): 37 °C, 1 h. Negative control (T−): No polymerase added to the mixtures or reactions with only dATP and dCTP or dATP, dCTP, and dTTP only. Positive control (T+): with all natural nucleotides and Taq polymerase. All reactions were incubated at adequate reaction temperatures for 1 h. P represents unreacted, 5′-FAM-labeled primer.

Fig. 4. Primer extension reactions with modified nucleotides 5 and 10 on a template containing a terminal stretch of dA nucleotides.

Gel image (PAGE 20%) shows the results of PEX reactions with 3′-phos-dTTP 5 and 3′-phos-LNA-TTP 10 with the P1/T2 primer/template system and various DNA polymerases. Modified triphosphates were at a final concentration of 200 µM. The following quantities of polymerases and reaction conditions were used: Phusion (2 U), Hemo Klen Taq (8 reactions), Taq (5 U), Bst (8 U), Q5 (2 U), Therminator (2 U), Vent (exo⁻) (2 U): 60 °C, 1 h; Dpo4 (2 U), Deep Vent (2 U): 55 °C, 1 h; Kf (exo⁻) (5 U): 37 °C, 1 h. Negative control (T−): No polymerase added. Positive control (T+): with all natural nucleotides and Taq polymerase. All reactions were incubated at adequate reaction temperatures for 1 h. P represents unreacted, 5′-FAM-labeled primer.

Fig. 5. Evaluation of modified nucleotide 5 under TdT-mediated reactions.

Gel image (PAGE 20%) shows the results of the TdT-mediated extension reactions with 3′-phos-dTTP 5 and 5′-FAM-labeled primer P2 (20 pmoles). Reaction mixtures contained TdT (10 U), triphosphate at 100 or 50 µM concentration, magnesium and/or manganese cofactors at 1 mM concentration, and were incubated at 37 °C for given reaction times. P represents unreacted, 5′-FAM-labeled primer.

Fig. 6. Synthesis of 3′-β-cyanophosphate-dTTP 11 and 3′-β-cyanophosphate-LNA-TTP 12.

A Synthetic scheme for the synthesis of nucleotide 11 and (B) synthetic scheme leading to nucleotide 12. Reagents and conditions: (i) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; 2. (nBu₃NH)₂ H₂P₂O₇, DMF, nBu₃N, rt, 45 min; 3. I₂, pyridine, H₂O, rt, 30 min, 13% over 3 steps for both 11 and 12.

Fig. 7. Evaluation of modified nucleotide 12 under TdT-mediated reactions.

Gel image (PAGE 20%) shows the results of the TdT-mediated extension reactions with 3′-β-cyanophosphate-LNA-TTP 12 and primer P2 (20 pmoles). Reaction mixtures contained TdT (10 U), triphosphate at various, given concentrations, Co²⁺ (0.25 mM) or Mn²⁺ (1 mM) cofactors, and were incubated at 37 °C for given reaction times. P represents unreacted, 5′-FAM-labeled primer.

Fig. 8. Synthesis of 3′-thiophosphate-LNA-TTP 15 and 3′-β-cyano-thiophosphate-LNA-TTP 16.

Reagents and conditions: (i) Beaucage reagent, pyridine, rt, 10 min, quantitative; (ii) TFA, DCM, rt, 60 min, quantitative; (iii) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; 2. (nBu₃NH)₂ H₂P₂O₇, DMF, nBu₃N, rt, 45 min; 3. I₂, pyridine, H₂O, rt, 30 min; 4. NH₄OH, MeNH₂ (1:1), rt, 2 h, 5% over 4 steps; iv) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; 2. (nBu₃NH)₂ H₂P₂O₇, DMF, nBu₃N, rt, 45 min; 3. I₂, pyridine, H₂O, rt, 30 min, 7% over 3 steps.

Fig. 9. Biochemical evaluation of nucleotide 15.

Gel image (PAGE 20%) shows the analysis of the products stemming from (A) PEX reactions with various DNA polymerases and the P1/T1 system and (B) TdT-mediated extension reactions with 5′-FAM-labeled primer P2. PEX reaction contained natural and modified triphosphates at 200 µM. The following quantities of polymerases were used: Phusion (2 U), Hemo Klen Taq (8 reactions), Taq (5 U), Bst (8 U), Q5 (2 U), Therminator (2 U), Vent (exo⁻) (2 U): 60 °C, 30 min; Dpo4 (2 U), Deep Vent (2 U): 55 °C, 30 min; Kf (exo⁻) (5 U): 37 °C, 30 min. Negative control (T−): No polymerase added to the mixtures or reactions with only dATP and dCTP or dATP, dCTP, and dTTP only. Positive control (T+): with all natural nucleotides and Taq polymerase. All reactions were incubated at adequate reaction temperatures for 1 h. TdT reaction mixtures contained TdT (10 U), triphosphate at various, given concentrations, Co²⁺ (0.25 mM) or Mn²⁺ (1 mM) cofactors, and were incubated at 37 °C for given reaction times. P represents unreacted, 5′-FAM-labeled primer.