3′‐O‐Caged 2′‐Deoxynucleoside Triphosphates for Light‐Mediated, Enzyme‐Catalyzed, Template‐Independent DNA Synthesis

Anu Stella Mathews1, Haikang Yang1, Carlo Montemagno1

1 Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.17
DOI:  10.1002/cpnc.41
Online Posting Date:  December, 2017
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Abstract

Synthesis, purification, and characterization of 3′‐O‐caged 2′‐deoxyribonucleoside triphosphates (dNTPs), namely 3′‐O‐(2‐nitrobenzyl)‐2′‐deoxy ribonucleoside triphosphates (NB‐dNTPs) and 3′‐O‐(4,5‐dimethoxy‐2‐nitrobenzyl)‐2′‐deoxy ribonucleoside triphosphates (DMNB‐dNTPs), are discussed in detail. A total of eight 3′‐O‐caged dNTPs are synthesized with specific protocols depending on the nitrogenous base on the first carbon, i.e., adenine, guanine, thymine, and cytosine, as well as the photo‐cleavable group, i.e, 2‐nitrobenzyl and 4,5‐ dimethoxy‐2‐nitrobenzyl, to be attached in the 3′‐O position. The purification of the synthesized compounds is done using ion‐exchange and flash chromatography; this is followed by structural confirmation by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The efficiency of the designed compounds is tested by conducting and evaluating UV‐cleaving experiments at 365 nm with proton NMR and LC‐MS curves. Finally, the application of the 3′‐O‐cagged dNTPs in template‐independent, enzyme‐catalyzed, photo‐mediated oligonucleotide synthesis is demonstrated. © 2017 by John Wiley & Sons, Inc.

Keywords: photo‐cleavable nucleotides; template independent DNA synthesis; terminal deoxynucleotidyl transferase; TdT enzyme

     
 
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Table of Contents

  • Introduction
  • Synthesis of 3′‐O‐Caged 2′‐Deoxyribonucleoside Triphosphates
  • Basic Protocol 1: Synthesis of 3′‐O‐(2‐Nitrobenzyl)‐2′‐Deoxyadenosine‐5′‐Triphosphate (NB‐dATP) and 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Deoxyadenosine‐5′‐Triphosphate (DMNB‐dATP)
  • Basic Protocol 2: Synthesis of 3′‐O‐(2‐Nitrobenzl)‐2′‐Deoxycytidine‐5′‐Triphosphate (NB‐dCTP) and Synthesis of 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Deoxycytidine‐ 5′‐Triphosphate (DMNB‐dCTP)
  • Basic Protocol 3: Synthesis of 3′‐O‐(2‐Nitrobenzyl)‐2′‐Deoxyguanosine‐5′‐Triphosphate (NB‐dGTP) and 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Deoxyguanosine‐5′‐ Triphosphate (DMNB‐dGTP)
  • Basic Protocol 4: Synthesis of 3′‐O‐(2‐Nitrobenzyl)‐2′‐Thymidine‐5′‐Triphosphate (NB‐dTTP) and 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Thymidine‐5′‐Triphosphate (DMNB‐dTTP)
  • Support Protocol 1: Photocleavability Studies of 3′‐O‐Caged 2′‐Deoxyribonucleoside Triphosphates
  • Basic Protocol 5: Template‐Independent Photo‐Mediated Enzymatic DNA Synthesis
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 3′‐O‐(2‐Nitrobenzyl)‐2′‐Deoxyadenosine‐5′‐Triphosphate (NB‐dATP) and 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Deoxyadenosine‐5′‐Triphosphate (DMNB‐dATP)

  Materials
  • 9‐[β‐D‐5′‐hydroxy‐2′‐deoxyribofuranosyl]‐6‐chloropurine (Berry & Associates, cat. no.PR3370)
  • Anhydrous N,N‐dimethylformamide (DMF; Sigma‐Aldrich, cat. no. SKU: 227056)
  • Imidazole (Sigma‐Aldrich, cat. no. SKU: 792527)
  • Argon
  • tert‐butyldimethylsilyl chloride (TBDMSCl; Aldrich, cat. no. SKU: 190500)
  • Ethyl acetate (EtOAc)
  • Brine (saturated aqueous NaCl)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel (230 to 400 mesh; Silicycle, cat. no. R10030B)
  • Hexane
  • Dichloromethane (DCM)
  • Tetrabutylammonium bromide (Sigma‐Aldrich, cat. no. SKU: 426288)
  • 2‐Nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 107794)
  • Sodium hydroxide (NaOH)
  • 4,5‐Dimethoxy‐2‐nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 392855)
  • Saturated sodium bicarbonate (NaHCO 3)
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich, cat. no. SKU: 401757)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF solution (Sigma‐Aldrich, cat. no. SKU: 216143)
  • Anhydrous 1,4‐dioxane (Sigma‐Aldrich, cat. no. SKU: 296309)
  • 7 N ammonia (NH 3) in methanol (Sigma‐Aldrich, cat. no. SKU: 499145)
  • Methanol (MeOH)
  • Anhydrous pyridine (Sigma‐Aldrich, cat. no. SKU: 270970)
  • 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one (Sigma‐Aldrich, cat. no. SKU: 324124)
  • Tributylammonium pyrophosphate (Sigma‐Aldrich, cat. no. SKU: P8533)
  • Tributylamine (Sigma‐Aldrich, cat. no. SKU: 90781)
  • Iodine (I 2)
  • Sodium sulfite (Na 2SO 3)
  • 1.0 M triethyl ammonium bicarbonate solution (TEAB; Sigma, cat. no. SKU: T7408)
  • DEAE‐Sephadex A‐25 (Sigma, cat. no. SKU: GE17‐0170‐01)
  • Nitrogen
  • 25‐, 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stir bars
  • Magnetic stirrer/hotplate
  • Rubber septa
  • Needles and syringes for adding reagents through septa
  • 250‐, 500‐, and 1000‐mL separatory funnels
  • 250‐mL Erlenmeyer flask
  • Fritted‐glass Büchner funnel setup with vacuum source
  • Rotary evaporator equipped with a coolant condenser and connected to an oil pump
  • 2.5 × 20‐ and 1.5 × 20‐cm glass chromatography columns
  • 250‐mL pressure flask (Chemglass, cat. no. SKU: CG‐1880‐R‐03)
  • Temperature‐controlled silicone/paraffin oil bath
  • Amber bottles
  • Additional reagents and equipment for column chromatography ( appendix 3E; Meyers, )

Basic Protocol 2: Synthesis of 3′‐O‐(2‐Nitrobenzl)‐2′‐Deoxycytidine‐5′‐Triphosphate (NB‐dCTP) and Synthesis of 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Deoxycytidine‐ 5′‐Triphosphate (DMNB‐dCTP)

  Materials
  • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyuridine (Alfa Aesar, cat. no. SKU: J65960)
  • Anhydrous N,N‐dimethylformamide (DMF; Sigma‐Aldrich, cat. no. SKU: 227056)
  • Imidazole (Sigma‐Aldrich, cat. no. SKU: 792527)
  • Argon
  • tert‐Butyldimethylsilyl chloride (TBDMSCl; Sigma‐Aldrich, SKU: 190500)
  • Ethyl acetate (EtOAc)
  • Hexanes
  • Brine (saturated aqueous NaCl)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel (230 to 400 mesh; Silicycle, cat. no. R10030B)
  • Dichloromethane (DCM)
  • Triethylamine (Sigma‐Aldrich, cat. no. SKU: T0886)
  • 4‐Dimethylaminopyridine (DMAP; Sigma‐Aldrich, cat. no. SKU: 107700)
  • 2‐Mesitylenesulfonyl chloride (Sigma‐Aldrich, cat. no. SKU: M7707)
  • Saturated sodium bicarbonate (NaHCO 3)
  • Allyl alcohol (Sigma‐Aldrich, cat. no. SKU: 240532)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Sigma‐Aldrich, cat. no. SKU: 139009)
  • Anhydrous magnesium sulfate (MgSO 4)
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich, SKU: 401757)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF solution (Sigma‐Aldrich, cat. no. SKU: 216143)
  • Tetrabutylammonium hydroxide (Bu 4NOH, 55% to 60% in water, Sigma‐Aldrich, SKU: 86863)
  • Sodium iodide (NaI)
  • Sodium hydroxide (NaOH)
  • 2‐Nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 107794)
  • 4,5‐Dimethoxy‐2‐nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 392855)
  • Trifluoroacetic acid (TFA, Sigma‐Aldrich, cat. no. SKU: T6508)
  • 7 N ammonia (NH 3; Sigma‐Aldrich, cat. no. SKU: 499145)) in methanol (MeOH)
  • Methanol (MeOH)
  • Anhydrous pyridine (Sigma‐Aldrich, cat. no. SKU: 270970)
  • 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one (Sigma‐Aldrich, cat. no. SKU: 324124)
  • Anhydrous 1,4‐dioxane (Sigma‐Aldrich, cat. no. SKU: 296309
  • Tributylammonium pyrophosphate (Sigma‐Aldrich, cat. no. SKU: P8533)
  • Tributylamine (Sigma‐Aldrich, cat. no. SKU: 90781)
  • Iodine (I 2)
  • Sodium sulfite (Na 2SO 3)
  • 1.0 M triethyl ammonium bicarbonate solution (TEAB; Sigma, cat. no. SKU: T7408)
  • DEAE‐Sephadex A‐25 (Sigma, cat. no. SKU: GE17‐0170‐01)
  • Nitrogen
  • 25‐, 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stir bars
  • Magnetic stirrer/hotplate
  • Rubber septa
  • Syringes and needles for adding reagents through septa
  • 250‐, 500‐, and 1000‐mL separatory funnels
  • 500‐ and 250‐mL Erlenmeyer flask
  • Fritted‐glass Büchner funnel setup with vacuum source
  • Rotary evaporator equipped with a coolant condenser and connected to an oil pump
  • 3.0 × 20‐, 2.5 × 20‐, and 1.5 × 20‐cm chromatography columns
  • Temperature‐controlled silicone/paraffin oil bath
  • 250‐mL pressure flask
  • Amber bottles
  • Additional reagents and equipment for column chromatography ( appendix 3E; Meyers, )

Basic Protocol 3: Synthesis of 3′‐O‐(2‐Nitrobenzyl)‐2′‐Deoxyguanosine‐5′‐Triphosphate (NB‐dGTP) and 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Deoxyguanosine‐5′‐ Triphosphate (DMNB‐dGTP)

  Materials
  • 2‐amino‐6‐chloro‐9‐[β‐D‐2′‐deoxyribofuranosyl]purine (Berry & Associates, cat. no. SKU: PR3040)
  • Anhydrous N,N‐dimethylformamide (DMF; Sigma‐Aldrich, cat. no. SKU: 227056)
  • tert‐Butyldimethylsilyl chloride (TBDMSCl; Sigma‐Aldrich, cat. no. SKU: 190500)
  • Imidazole (Sigma‐Aldrich, cat. no. SKU: 792527)
  • Argon
  • Ethyl acetate (EtOAc)
  • Brine (saturated aqueous NaCl)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Hexane
  • Silica gel (230 to 400 mesh; Silicycle, cat. no. R10030B)
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich, cat. no. SKU: 401757)
  • N,N‐dimethylformamide dimethylacetal (Sigma‐Aldrich, cat. no. SKU: 140732)
  • Dichloromethane (DCM)
  • Methanol (MeOH)
  • 95% sodium hydride (NaH; Sigma‐Aldrich, cat. no. SKU: 223441)
  • 2‐Nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 107794)Saturated sodium bicarbonate (NaHCO 3)
  • 4,5‐dimethoxy‐2‐nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 392855)
  • Cesium acetate (Sigma‐Aldrich, cat. no. SKU: 441902)
  • 1,4‐diazabicyclo[2.2.2]octane (DABCO, Sigma‐Aldrich, cat. no. SKU: D27802))
  • Triethylamine (Sigma‐Aldrich, cat. no. SKU: T0886)
  • Acetic anhydride (Ac 2O; Sigma‐Aldrich, cat. no. SKU: 320102)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF solution (Sigma‐Aldrich, cat. no. SKU: 216143)
  • Anhydrous pyridine (Sigma‐Aldrich, cat. no. SKU: 270970)
  • 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one (Sigma‐Aldrich, cat. no. SKU: 324124)
  • Anhydrous 1,4‐dioxane (Sigma‐Aldrich, cat. no. SKU: 296309
  • Tributylammonium pyrophosphate (Sigma‐Aldrich, cat. no. SKU: P8533)
  • Tributylamine (Sigma‐Aldrich, cat. no. SKU: 90781)
  • Iodine (I 2)
  • Sodium sulfite (Na 2SO 3)
  • 1.0 M triethyl ammonium bicarbonate solution (TEAB; Sigma, cat. no. SKU: T7408)
  • Concentrated aqueous ammonium hydroxide (NH 4OH)
  • DEAE‐Sephadex A‐25 (Sigma, cat. no. SKU: GE17‐0170‐01)
  • 50‐, 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stir bars
  • Magnetic stirrer/hotplate
  • Rubber septa
  • Syringes and needles for adding reagents through septa
  • Nitrogen
  • 125‐, 250‐, 500‐, and 1000‐mL separatory funnels
  • Fritted‐glass Büchner funnel setup with vacuum source
  • Rotary evaporator equipped with a coolant condenser and connected to an oil pump
  • 2.5 × 20‐ and 1.5 × 20‐cm glass chromatography column
  • Cooling condensers
  • 250‐ and 500‐mL Erlenmeyer flasks
  • Temperature‐controlled silicone/paraffin oil bath
  • Reflux condensers
  • Amber bottles
  • Additional reagents and equipment for column chromatography ( appendix 3E; Meyers, )

Basic Protocol 4: Synthesis of 3′‐O‐(2‐Nitrobenzyl)‐2′‐Thymidine‐5′‐Triphosphate (NB‐dTTP) and 3′‐O‐(4,5‐Dimethoxy‐2‐Nitrobenzyl)‐2′‐Thymidine‐5′‐Triphosphate (DMNB‐dTTP)

  Materials
  • 5′‐O‐(tert‐butyldimethyl)thymidine (Berry & Associates, cat. no. SKU: PY7140)
  • Anhydrous dichloromethane (DCM, Sigma‐Aldrich, cat. no. SKU: 270997)
  • Triethylamine (Sigma‐Aldrich, cat. no. SKU: T0886)
  • Benzoyl chloride (Sigma‐Aldrich, cat. no. SKU: 259950)
  • Argon
  • Brine (saturated aqueous NaCl)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Tetrabutylammonium hydroxide (Bu 4NOH, 55° to 60% in water;, Sigma‐Aldrich, cat. no. SKU: 86863)
  • Sodium iodide (NaI)
  • Sodium hydroxide (NaOH)2‐nitrobenzyl bromide (Sigma‐Aldrich, cat. no. SKU: 107794)
  • Silica gel (230 to 400 mesh; Silicycle, cat. no. R10030B)
  • Hexanes
  • Ethyl acetate (EtOAc)4,5‐dimethoxy‐2‐nitrobenzyl bromide (Aldrich, cat. no. SKU: 392855)
  • Ethanol (EtOH)
  • Concentrated ammonium hydroxide (NH 4OH)
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich, cat. no. SKU: 401757)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF solution (Aldrich, cat. no. SKU: 216143)
  • Methanol (MeOH)
  • Anhydrous N,N‐dimethylformamide (DMF, Sigma‐Aldrich, cat. no. SKU: 227056)
  • Anhydrous pyridine (Sigma‐Aldrich, cat. no. SKU: 270970)
  • 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one (Sigma‐Aldrich, cat. no. SKU: 324124)
  • Anhydrous 1,4‐dioxane (Sigma‐Aldrich, cat. no. SKU: 296309
  • Tributylammonium pyrophosphate (Aldrich, cat. no. SKU: P8533)
  • Tributylamine (Sigma‐Aldrich, cat. no. SKU: 90781)
  • Iodine (I 2)
  • Sodium sulfite (Na 2SO 3)
  • 1.0 M triethyl ammonium bicarbonate solution (TEAB, cat. no. Sigma, SKU: T7408)
  • DEAE‐Sephadex A‐25 (Sigma, cat. no. SKU: GE17‐0170‐01)
  • Nitrogen
  • 50‐, 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stir bars
  • Magnetic stirrer/hotplate
  • 125‐, 250‐, 500‐, and 1000‐mL separatory funnels
  • Fritted‐glass Büchner funnel setup with vacuum source
  • Rotary evaporator equipped with coolant condenser and connected to an oil pump
  • 2.5 × 20‐ and 1.5 × 20‐cm glass chromatography columns
  • Amber bottles
  • Additional reagents and equipment for column chromatography ( appendix 3E; Meyers, )

Support Protocol 1: Photocleavability Studies of 3′‐O‐Caged 2′‐Deoxyribonucleoside Triphosphates

  Materials
  • 3′‐O‐caged dNTPs (Basic Protocols protocol 11 to protocol 44)
  • Deuterium oxide (D 2O)
  • NMR tubes
  • UV lamp (ORIEL instruments, Model 67005)
  • UV filter (THORLABS, cat no. FGUV5S)

Basic Protocol 5: Template‐Independent Photo‐Mediated Enzymatic DNA Synthesis

  Materials
  • DNA (92.5 ng/μL, 750 bp)
  • 5× TdT buffer (Thermo Scientific, cat. no. PN72035)
  • 100 mM CoCl 2 solution
  • 10 mM dNTP mix: commercial non‐caged dNTP and/or caged dNTPs
  • 20 U/µl terminal deoxynucleotidyl transferase, (TdT; Thermo Scientific, cat. no. PN72033)
  • Oligonucleotide (d(CCCAGTCACGACGT OH)) attached to 1 micromole synthesis column, 1000A(MM1‐1100A‐1, Bioautomation)
  • 10 mM NB‐dNTP (Basic Protocols protocol 11 to protocol 44)
  • UV light source
  • Mermade 192 automated oligonucleotide synthesizer (BioAutomation)
  • Controlled pore glass (CPG) column (50 nm pore size)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ), oligonucleotide synthesis and purification ( appendix 3c; Ellington & Pollard, ), and MALDI‐TOF mass spectrometry of oligonucleotides (unit 10.1; Castleberry et al., )
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Literature Cited

Literature Cited
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  Ellington, A., & Pollard, J. D. (2001). Introduction to the synthesis and purification of oligonucleotides. Current Protocols in Nucleic Acid Chemistry, 00, A.3C.1–A.3C.22. doi: 10.1002/0471142700.nca03cs00.
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