3'-O-reversibly closed nucleotide and application thereof in template-free enzymatic nucleic acid synthesis

Abstract

The invention relates to the field of enzymatic nucleic acid synthesis, and particularly provides 3'-O-reversibly closed nucleotide and application thereof in template-free enzymatic nucleic acid synthesis. According to the 3'-O-reversibly closed nucleotide provided by the invention, a closing group is cyano vinyl; the cyano vinyl has the effect of realizing reversible closing of 3'-OH of the nucleotide; the cyano vinyl is relatively small in volume, relatively stable in structure, easy to synthesize and low in cost; meanwhile, the cyano vinyl can generate hydrogen bond interaction with a TdT enzyme catalytic site to stably bind conformation; the TdT enzyme catalytic reaction is very facilitated; and the reaction rate of enzymatic nucleic acid synthesis is improved.

Description

technical field

[0001] The invention relates to the field of enzymatic nucleic acid synthesis, in particular to a 3'-O-reversibly blocked nucleotide and its application in template-free enzymatic nucleic acid synthesis. Background technique

[0002] The currently widely used oligonucleotide synthesis method is based on the chemical synthesis technology of solid-phase phosphoramidite "four-step method of deprotection, coupling, capping and oxidation". However, due to the limitations of synthetic chemistry technology, its limit synthesis length is about 200 nt, which is difficult to fully meet the needs of synthetic biology and DNA storage technology for high-throughput, high-fidelity, long-fragment, and low-cost DNA synthesis, and further development is needed New Enzymatic DNA Synthesis Technology.

[0003] In DNA enzymatic synthesis, TdT enzyme (DNA terminal transferase, Terminal deoxynucleotidyltransferase) is a DNA polymerase that can catalyze the binding of deoxyribonuc...

Examples

Embodiment 1

[0057] Embodiment 1, TdT enzymatic DNA extension experiment

[0058] (1) Experimental materials

[0059] 1. OligoDNA sequence: 5'-GCAGA TAATA CGACT CACTA TAGGG ATTTA GACTA CCCCAAAAAC GAAGG GGACT AAAAC-3' (60nt, SEQ ID NO.2);

[0060] 2. TdT (0.12mg / mL) (SEQ ID NO.1);

[0061] 3. TdT reaction buffer: 10×TdT reaction buffer, 10×CoCl 2 (2.5mM) (NEB, B0315S), where 1×TdT buffer: 20mM Tris-Acetate, 50mM KAc, 10mM MgAc 2 ,pH 7.9@25℃;

[0062] 4. Substrate: 3'-O-cyanovinyl-dTTP (CTTP, 10mM), 3'-O-Azidomethyl-dTTP (ATTP, 10mM), dNTP mix (dNTP, 10mM);

[0063] 5. ddH 2 O;

[0064] (2) Experimental operation

[0065] (1) Configure four TdT enzymatic DNA extension reaction systems:

[0066] oligoDNA (5μL, 2.5mM, 60nt), 10×TdT reaction buffer (5μL), 2.5mM CoCl 2 Solution (5μL), 8.3μL TdT (0.12mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5mL), and ddH 2 O dilute the mixture to 50 μL;

[0067]

[0068]

[0069] (2) with ddH 2 O...

Embodiment 2

[0075] Embodiment 2, deprotection experiment

[0076] (1) Experimental materials

[0077] Deprotection reagents: 0.5M TCEP (pH=10) solution for ATTP substrate, 0.05M diacetonitrile palladium dichloride (PdCl 2 (CH 3 EN) 2 ) solution is used for CTTP substrate;

[0078] (2) Experimental operation

[0079] (1) Configure four TdT enzymatic DNA extension reaction systems:

[0080] oligoDNA (15μL, 2.5mM, 60nt), 10×TdT reaction buffer (15μL), 2.5mM CoCl 2 Solution (15μL), 16.6μL TdT (0.12mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5mL), and washed with ddH 2 O dilute the mixture to 150 μL;

[0081] # Substrate and system Volume (μL) 1 CTTP 1.5 2 ATTP 1.5 3 dNTP 12 4 blank --(no added)

[0082] (2) The mixture was incubated at 37°C for 1 hour, 4.5 hours and 5 hours according to different substrates;

[0083] (3) Take out the reaction solution, add 2.5 μL 0.5M TCEP (pH=10) solution to...

Embodiment 3、3

[0090] The synthesis of embodiment 3,3'-O-cyanovinyl-dNTP

[0091] Using 5'-OTBDMS-protected nucleosides as raw materials, firstly through a condensation reaction, the 3'-OH is selectively directly esterified with formic acid to generate the corresponding methyl ester. Utilizing the wittig reactivity of methyl ester, react with phosphorus ylide reagent (triphenylphosphoranylidene) acetonitrile to generate 3'-O-cyanovinyl, and obtain 3'-O-cyanovinyl after removal of 5'-OTBDMS -dNTP precursor, followed by triphosphorylation and deprotection to synthesize the desired substrate. Its specific synthetic flow chart is as Figure 5 Shown ((i) HCOOH, EDCI, DMAP, DCM, 0℃-room temperature, 6h; (ii) (1) 2-(triphenylphosphaneylidene) acetonitrile (Wittigreagents), Toluene, 120℃, 9h; (2) TBAF·3H 2 O, THF, room temperature, 1h; (iv) (1) POCl 3 ,1,8-Bis(dimethyl amino)naphthalene,(MeO) 3 PO, 0℃, 2h; (2) Tributylammoniumpyrophosphate, Bu 3 N,CH 3 CN, 0℃, 10min; (3)NH 4 OH, 25°C, 16h). ...

technical field

[0001] The invention relates to the field of enzymatic nucleic acid synthesis, in particular to a 3'-O-reversibly blocked nucleotide and its application in template-free enzymatic nucleic acid synthesis. Background technique

[0002] The currently widely used oligonucleotide synthesis method is based on the chemical synthesis technology of solid-phase phosphoramidite "four-step method of deprotection, coupling, capping and oxidation". However, due to the limitations of synthetic chemistry technology, its limit synthesis length is about 200 nt, which is difficult to fully meet the needs of synthetic biology and DNA storage technology for high-throughput, high-fidelity, long-fragment, and low-cost DNA synthesis, and further development is needed New Enzymatic DNA Synthesis Technology.

[0003] In DNA enzymatic synthesis, TdT enzyme (DNA terminal transferase, Terminal deoxynucleotidyltransferase) is a DNA polymerase that can catalyze the binding of deoxyribonuc...

Examples

Embodiment 1

[0057] Embodiment 1, TdT enzymatic DNA extension experiment

[0058] (1) Experimental materials

[0059] 1. OligoDNA sequence: 5'-GCAGA TAATA CGACT CACTA TAGGG ATTTA GACTA CCCCAAAAAC GAAGG GGACT AAAAC-3' (60nt, SEQ ID NO.2);

[0060] 2. TdT (0.12mg / mL) (SEQ ID NO.1);

[0061] 3. TdT reaction buffer: 10×TdT reaction buffer, 10×CoCl 2 (2.5mM) (NEB, B0315S), where 1×TdT buffer: 20mM Tris-Acetate, 50mM KAc, 10mM MgAc 2 ,pH 7.9@25℃;

[0062] 4. Substrate: 3'-O-cyanovinyl-dTTP (CTTP, 10mM), 3'-O-Azidomethyl-dTTP (ATTP, 10mM), dNTP mix (dNTP, 10mM);

[0063] 5. ddH 2 O;

[0064] (2) Experimental operation

[0065] (1) Configure four TdT enzymatic DNA extension reaction systems:

[0066] oligoDNA (5μL, 2.5mM, 60nt), 10×TdT reaction buffer (5μL), 2.5mM CoCl 2 Solution (5μL), 8.3μL TdT (0.12mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5mL), and ddH 2 O dilute the mixture to 50 μL;

[0067]

[0068]

[0069] (2) with ddH 2 O...

Embodiment 2

[0075] Embodiment 2, deprotection experiment

[0076] (1) Experimental materials

[0077] Deprotection reagents: 0.5M TCEP (pH=10) solution for ATTP substrate, 0.05M diacetonitrile palladium dichloride (PdCl 2 (CH 3 EN) 2 ) solution is used for CTTP substrate;

[0078] (2) Experimental operation

[0079] (1) Configure four TdT enzymatic DNA extension reaction systems:

[0080] oligoDNA (15μL, 2.5mM, 60nt), 10×TdT reaction buffer (15μL), 2.5mM CoCl 2 Solution (15μL), 16.6μL TdT (0.12mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5mL), and washed with ddH 2 O dilute the mixture to 150 μL;

[0081] # Substrate and system Volume (μL) 1 CTTP 1.5 2 ATTP 1.5 3 dNTP 12 4 blank --(no added)

[0082] (2) The mixture was incubated at 37°C for 1 hour, 4.5 hours and 5 hours according to different substrates;

[0083] (3) Take out the reaction solution, add 2.5 μL 0.5M TCEP (pH=10) solution to...

Embodiment 3、3

[0090] The synthesis of embodiment 3,3'-O-cyanovinyl-dNTP

[0091] Using 5'-OTBDMS-protected nucleosides as raw materials, firstly through a condensation reaction, the 3'-OH is selectively directly esterified with formic acid to generate the corresponding methyl ester. Utilizing the wittig reactivity of methyl ester, react with phosphorus ylide reagent (triphenylphosphoranylidene) acetonitrile to generate 3'-O-cyanovinyl, and obtain 3'-O-cyanovinyl after removal of 5'-OTBDMS -dNTP precursor, followed by triphosphorylation and deprotection to synthesize the desired substrate. Its specific synthetic flow chart is as Figure 5 Shown ((i) HCOOH, EDCI, DMAP, DCM, 0℃-room temperature, 6h; (ii) (1) 2-(triphenylphosphaneylidene) acetonitrile (Wittigreagents), Toluene, 120℃, 9h; (2) TBAF·3H 2 O, THF, room temperature, 1h; (iv) (1) POCl 3 ,1,8-Bis(dimethyl amino)naphthalene,(MeO) 3 PO, 0℃, 2h; (2) Tributylammoniumpyrophosphate, Bu 3 N,CH 3 CN, 0℃, 10min; (3)NH 4 OH, 25°C, 16h). ...

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