Synthesis of Threose Nucleic Acid (TNA) Triphosphates and Oligonucleotides by Polymerase‐Mediated Primer Extension

Su Zhang1, Hanyang Yu1, John C. Chaput1

1 Center for Evolutionary Medicine and Informatics, The Biodesign Institute, and Department of Chemistry and Biochemistry at Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.54
DOI:  10.1002/0471142700.nc0454s52
Online Posting Date:  March, 2013
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Abstract

This unit describes the chemical synthesis of α‐L‐threofuranosyl nucleic acid (TNA) triphosphates for thymidine (T), guanosine (G), cytidine (C), and the diaminopurine (D) analog of adenosine and their incorporation into TNA oligonucleotides by enzyme‐mediated polymerization of a DNA primer‐template complex. Starting from suitably protected threofuranosyl nucleosides, TNA triphosphates are synthesized in a single‐pot reaction and purified by ion‐exchange and HPLC chromatography. Purified TNA triphosphates are diluted into stock solutions and used as substrates for the synthesis of TNA oligonucleotides. Oligonucleotide synthesis is accomplished using Therminator DNA polymerase, a commercial variant of the 9oN DNA polymerase bearing the A485L mutation. Curr. Protoc. Nucleic Acid Chem. 52:4.54.1‐4.54.17. © 2013 by John Wiley & Sons, Inc.

Keywords: alternative nucleic acids; threose nucleic acid (TNA); triphosphates; oligonucleotide; polymerase‐mediated primer extension

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of (α‐L‐Threofuranosyl)Cytosine‐3′‐Triphosphate
  • Basic Protocol 2: Enzymatic Synthesis of Threose Nucleic Acid (TNA) Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 1‐{2′‐O‐[(4′′,4′′′‐dimethoxytriphenyl)methyl]‐α‐L‐threofuranosyl}thymine
  • Pyridine, freshly distilled
  • Phosphorous pentoxide (P 2O 5)
  • Argon balloons
  • 1,4‐Dioxane, freshly distilled
  • 2‐Chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one
  • Tributylammonium pyrophosphate
  • N,N‐Dimethyformamide, freshly distilled (DMF)
  • Tributylamine, freshly distilled
  • 1% I 2 solution in 98:2 (v/v) pyridine/water
  • 5% aq. Na 2SO 3
  • 50% aq. acetic acid
  • Diethyl ether (Et 2O)
  • 1 M aq. triethylammonium bicarbonate buffer, pH 8.0 (TEAB)
  • 100 mM aq. triethylammonium acetate buffer, pH 7.0 (TEAA)
  • Methanol (HPLC‐grade MeOH)
  • Acetone, HPLC grade
  • Sodium perchlorate (NaClO 4)
  • 10‐ and 50‐mL round‐bottom flasks, oven‐dried
  • 50‐mL separatory funnel
  • Rotary evaporator equipped with a vacuum pump
  • Magnetic stir plate and stir bar
  • Vacuum desiccator
  • Lyophilizer
  • GE Pharmacia ÄKTA FPLC system
  • Customized DEAE Sephadex anion‐exchange column (Essential Life Solutions)
  • ZORBAX C‐18 reversed‐phase HPLC column (Agilent Technologies)
  • Agilent 1100 HPLC system
  • Additional reagents and equipment for HPLC (unit 10.5)

Basic Protocol 2:

  Materials
  • N2,N6‐Dibenzoyl‐9‐{2′‐O‐[(4′′,4′′′‐dimethoxytriphenyl)methyl]‐α‐L‐threofuranosyl}‐2,6‐diaminopurine
  • 8 M methylamine in ethanol
  • 12 M methylamine in water
  • Dichloromethane (CH 2Cl 2)
  • Triethylamine (Et 3N)
  • Methanol, HPLC grade
  • Pyridine, freshly distilled
  • Phosphorous pentoxide (P 2O 5)
  • Argon balloons
  • N,N‐dimethyformamide (freshly distilled DMF)
  • 2‐Chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one
  • 1,4‐Dioxane, freshly distilled
  • Tributylammonium pyrophosphate
  • Tributylamine, freshly distilled
  • 1% I 2 solution in 98:2 pyridine/water
  • 5% aq. Na 2SO 3
  • 50% aq. acetic acid
  • Diethyl ether (Et 2O)
  • 1 M aq. triethylammonium bicarbonate buffer, pH 8.0 (TEAB)
  • 100 mM aq. triethylammonium acetate buffer, pH 7.0 (TEAA)
  • Acetone, HPLC grade
  • Sodium perchlorate (NaClO 4)
  • Magnetic stir plate and stir bar
  • 10‐ and 50‐mL round‐bottom flasks
  • Rotary evaporator equipped with a vacuum pump
  • 6.4 × 45‐cm chromatography column
  • 50‐mL separatory funnel
  • Vacuum desiccators
  • Lyophilizer
  • Customized DEAE Sephadex anion‐exchange column (Essential Life Solutions)
  • GE Pharmacia ÄKTA FPLC system
  • ZORBAX C‐18 reversed‐phase HPLC column (Agilent Technologies)
  • Agilent 1100 HPLC system
  • Additional reagents and equipment for column chromatography ( appendix 3E), and HPLC (unit 10.5)

Basic Protocol 3:

  Materials
  • N2‐Acetyl‐O6‐diphenylcarbamoyl‐9‐{2′‐O‐[(4′′,4′′′‐dimethoxytriphenyl)methyl]‐α‐L‐threofuranosyl}‐guanine
  • 8 M methylamine in ethanol
  • 12 M methylamine in water
  • Dichloromethane (CH 2Cl 2)
  • Triethylamine (Et 3N)
  • Methanol (HPLC‐grade MeOH)
  • Pyridine, freshly distilled
  • Phosphorous pentoxide (P 2O 5)
  • Argon balloons
  • N,N‐Dimethyformamide, freshly distilled (DMF)
  • 2‐Chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one
  • 1,4‐Dioxane, freshly distilled
  • Tributylammonium pyrophosphate
  • Tributylamine, freshly distilled
  • 1% I 2 solution in 98:2 pyridine/water
  • 5% aq. Na 2SO 3
  • 50% aq. acetic acid
  • Diethyl ether (Et 2O)
  • 1 M aq. triethylammonium bicarbonate buffer, pH 8.0 (TEAB)
  • 100 mM aq. triethylammonium acetate buffer, pH 7.0 (TEAA)
  • Acetone, HPLC grade
  • Sodium perchlorate (NaClO 4)
  • Magnetic stir plate and stir bar
  • 10‐ and 50‐mL round‐bottom flasks
  • Rotary evaporator equipped with a vacuum pump
  • 6.4 × 45‐cm chromatography column
  • 50‐mL separatory funnel
  • Vacuum desiccator
  • Lyophilizer
  • Customized DEAE Sephadex anion‐exchange column (Essential Life Solutions)
  • GE Pharmacia ÄKTA FPLC system
  • ZORBAX C‐18 reversed‐phase HPLC column (Agilent Technologies)
  • Agilent 1100 HPLC system
  • Additional reagents and equipment for thin layer chromatography (TLC) ( appendix 3D), column chromatography ( appendix 3E), and HPLC (unit 10.5)

Basic Protocol 4: Synthesis of (α‐L‐Threofuranosyl)Cytosine‐3′‐Triphosphate

  Materials
  • N4‐Benzoyl‐1‐{3′‐O‐[(4′′,4′′′‐dimethoxytriphenyl)methyl]‐α‐L‐threofuranosyl}cytosine(9)
  • Pyridine, freshly distilled
  • Acetic anhydride
  • Argon balloons
  • Methanol (HPLC‐grade MeOH)
  • Dichloromethane (CH 2Cl 2)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Hexanes
  • Triethylamine (Et 3N)
  • Trichloroacetic acid (TCA)
  • SiO 2
  • Phosphorous pentoxide (P 2O 5)
  • 1,4‐Dioxane, freshly distilled
  • 2‐Chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one
  • Tributylammonium pyrophosphate
  • N,N‐Dimethyformamide, freshly distilled (DMF)
  • Tributylamine, freshly distilled
  • 1% I 2 solution in 98:2 pyridine/water
  • 5% aq. Na 2SO 3
  • Concentrated (28%) ammonium hydroxide (NH 4OH)
  • Diethyl ether (Et 2O)
  • 1 M aq. triethylammonium bicarbonate buffer, pH 8.0 (TEAB)
  • 100 mM aq. triethylammonium acetate buffer, pH 7.0 (TEAA)
  • Acetone, HPLC grade
  • Sodium perchlorate (NaClO 4)
  • Magnetic stir plate and stir bar
  • 10‐ and 50‐mL round‐bottom flasks, oven dried
  • Rotary evaporator equipped with a vacuum pump
  • 50‐ and 250‐ml separatory funnels
  • Vacuum desiccator
  • Lyophilizer
  • Customized DEAE Sephadex anion‐exchange column (Essential Life Solutions)
  • GE Pharmacia ÄKTA FPLC system
  • ZORBAX C‐18 reversed‐phase HPLC column (Agilent Technologies)
  • Agilent 1100 HPLC system
  • Additional reagents and equipment for thin layer chromatography (TLC; appendix 3D), column chromatography ( appendix 3E), and HPLC (unit 10.5)

Basic Protocol 5: Enzymatic Synthesis of Threose Nucleic Acid (TNA) Oligonucleotides

  Materials
  • 5 mM (α‐L‐threofuranosyl)thymine‐3′‐triphosphate (tTTP)
  • 5 mM (α‐L‐threofuranosyl)‐2,6‐diaminopurine‐3′‐triphosphate (tDTP)
  • 5 mM (α‐L‐threofuranosyl)guanine‐3′‐triphosphate (tGTP)
  • 5 mM (α‐L‐threofuranosyl)cytosine‐3′‐triphosphate (tCTP)
  • MnCl 2·4H 2O (mol. wt. 197.91 g/mol, Fisher)
  • Dithiothreitol (DTT, mol. wt. 154.3 g/mol, BioRad)
  • 5 µM DNA primer
  • 10 µM DNA template
  • 10× ThermoPol reaction buffer (New England BioLabs)
  • 10 mg/mL BSA (New England BioLabs)
  • Therminator DNA polymerase (2000 U/mL, New England BioLabs)
  • Microcentrifuge tubes
  • 95°C heating block
  • 55°C water bath
  • UV spectrophotometer
  • Additional reagents and equipment for denaturing PAGE and ethanol precipitation (unit 10.4)
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Figures

Videos

Literature Cited

Literature Cited
   Chaput, J.C. and Szostak, J.W. 2003. TNA synthesis by DNA polymerases. J. Am. Chem. Soc. 125:9274‐9275.
   Chaput, J.C., Ichida, J.K., and Szostak, J.W. 2003. DNA polymerase‐mediated DNA synthesis on a TNA template. J. Am. Chem. Soc. 125:856‐857.
   Cooper, G., Kimmich, N., Belisle, W., Sarinana, J., Brabham, K., and Garrel, L. 2001. Carbonaceous meteorites as a source of sugar‐related organic compounds for the early earth. Nature 414:879‐883.
   Ebert, M.‐O., Mang, C., Krishnamurthy, R., Eschenmoser, A., and Jaun, B. 2008. The structure of a TNA‐TNA complex in solution: NMR study of the octamer duplex derived from α‐(L)‐threofuranosyl‐(3′‐2′)‐CGAATTCG. J. Am. Chem. Soc. 130:15105‐15115.
   Eschenmoser, A. 1999. Chemical etiology of nucleic acid structure. Science 284:2118‐2124.
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   Horhota, A., Zou, K., Ichida, J.K., Yu, B., McLaughlin, L.W., Szostak, J.W., and Chaput, J.C. 2005. Kinetic analysis of an efficient DNA‐dependent TNA polymerase. J. Am. Chem. Soc. 127:7427‐7434.
   Ichida, J.K., Horhota, A., Zou, K., McLaughlin, L.W., and Szostak, J.W. 2005a. High fidelity TNA synthesis by Therminator polymerase. Nucleic Acids Res. 33:5219‐5225.
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   Pinheiro, V.B., Taylor, A.I., Cozens, C., Abramov, M., Renders, M., Zhang, S., Chaput, J.C., Wengel, J., Peak‐Chew, S.‐Y., McLaughlin, S.H., Herdewijn, P., and Holliger, P. 2012. Synthetic genetic polymers capable of heredity and evolution. Science 336:341‐344.
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   Schöning, K.‐U., Scholz, P., Wu, X., Guntha, S., Delgado, G., Krishnamurthy, R., and Eschenmoser, A. 2002. The α‐L‐threofuranosyl‐(3′→2′)‐oligonucleotide system (‘TNA’): Synthesis and pairing properties. Helv. Chim. Acta 85:4111‐4153.
   Wilds, C.J., Warwrzak, Z., Krishnamurthy, R., Eschenmoser, A., and Egli, M. 2002. Crystal structure of a B‐form DNA duplex containing (L)‐α‐threofuranosyl (3′→2′) nucleosides: A four‐carbon sugar is easily accommodated into the backbone of DNA. J. Am. Chem. Soc. 124:13716‐13721.
   Yang, Y.‐W., Zhang, S., McCullum, E.O., and Chaput, J.C. 2007. Experimental evidence that GNA and TNA were not sequential polymers in the prebiotic evolution of RNA. J. Mol. Evol. 65:289‐295.
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