Nonenzymatic Oligomerization of Activated Nucleotides on Hairpin Templates

Eun‐Kyong Kim1, Christopher Switzer1

1 Department of Chemistry, University of California, Riverside, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.18
DOI:  10.1002/0471142700.nc0318s39
Online Posting Date:  December, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a protocol for nonenzymatic oligomerization of activated ribonucleotides on DNA hairpins appended by templates containing threofuranosyl nucleic acid (TNA). TNA‐cytidylate templates effectively promote oligomerization of 2‐MeImpG, and give 3′,5′‐linked oligomerization products predominantly, with good base‐pairing fidelity. Although the rates of oligomerization depend on TNA content, after 3 days of incubation, oligomerization products are apparent, and full‐length products are present after 10 days. Characterization of product phosphodiester bond regiochemistry is accomplished by digestion with RNase T1. Additionally, exposure of oligomerization products to calf intestinal alkaline phosphatase enables detection of any endcapping due to pyrophosphate formation. Base‐pairing fidelity is assessed by challenging the template to oligomerize 2‐MeImpA. The protocols described for nonenzymatic, template‐directed synthesis in this unit are applicable to oligomerization of activated monomers on templates of different compositions, with respect to both base identity and polymer backbone. Curr. Protoc. Nucleic Acid Chem. 39:3.18.1‐3.18.13. © 2009 by John Wiley & Sons, Inc.

Keywords: nonenzymatic; template‐directed; oligomerization; RNA; DNA; TNA; prebiotic

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Activated Nucleotide Monomers
  • Basic Protocol 2: Nonenzymatic Oligomerization of RNA by TNA Templates
  • Support Protocol 1: Synthesis of TNA‐Bearing Hairpin Oligonucleotides
  • Alternate Protocol 1: Base‐Pairing Fidelity and Effects of Different Divalent Ions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Activated Nucleotide Monomers

  Materials
  • Adenosine 5′‐monophosphate (AMP; Sigma)
  • Guanosine 5′‐monophosphate (GMP; Sigma)
  • 2‐methylimidazole (2‐MeIm), 99% (Aldrich)
  • Dimethyl sulfoxide (DMSO), distilled from CaH 2
  • Nitrogen (or argon) gas, dry
  • N,N‐Dimethylformamide (DMF), water < 50 ppm (Acros Organics)
  • Triphenylphosphine (Ph 3P), 99% (Aldrich)
  • 2,2′‐Dipyridyl disulfide (PySSPy, AldrithiolT‐2), 98% (Aldrich)
  • Triethylamine (Et 3N, TEA), distilled from KOH and stored over KOH
  • 11:2:7 (v/v/v) n‐propanol/ammonium hydroxide/water
  • Acetone (Fisher Scientific)
  • Diethyl ether, anhydrous (Fisher Scientific)
  • Saturated anhydrous sodium perchlorate (NaClO 4; Aldrich) in acetone
  • Phosphorus pentoxide (P 2O 5), anhydrous, 99+% (Acros Organics)
  • Sodium hydroxide (NaOH), certified ACS (Fisher scientific)
  • 50‐mL round‐bottom flasks
  • Magnetic stir bars and plate
  • TLC silica gel plates with UV indicator, Merck silica gel 60 F254
  • Benchtop centrifuge
  • Desiccator
  • High vacuum oil pump
  • Additional reagents and equipment for performing TLC ( appendix 3D)

Basic Protocol 2: Nonenzymatic Oligomerization of RNA by TNA Templates

  Materials
  • Templates: Hairpin oligonucleotides bearing TNA‐C residues ( protocol 3)
  • T4 polynucleotide kinase and dilution buffer (USB)
  • 10× kinase buffer; 5:1:1:3 (v/v/v/v) 1.4 M Tris⋅Cl, pH 7.6/1.0 M MgCl 2/500 mM dithiothreitol (DTT)/H 2O
  • [γ‐32P]ATP, 6000 Ci/mmol, 10 mCi/mL (Perkin‐Elmer & Analytical Science)
  • 660 µM ATP
  • Loading buffer (7 M urea in 1× TBE buffer containing 0.1 % xylene cyanol and 0.1 % bromphenol blue)
  • 7 M urea/20% denaturing polyacrylamide gel
  • 50 mM TEAA buffer, pH 7.0 (see recipe)
  • 2 M NaCl
  • 1 M MgCl 2
  • 1 M 2,6‐lutidine⋅HCl, pH 8.0 (see recipe)
  • 2‐MeImpG ( protocol 1)
  • 2‐MeImpA ( protocol 1)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • Ribonuclease T1 (RNase T1; Boehringer Mannheim)
  • Tris‐EDTA buffer (10 mM Tris⋅Cl, pH 7.4/1 mM EDTA, pH 8.0)
  • Calf intestinal alkaline phosphatase (CIAP)
  • 10× phosphatase buffer (20 mM Tris⋅Cl, pH 8.0/10 mM MgCl 2)
  • 1.5‐mL microcentrifuge tubes
  • Vortex mixer
  • Benchtop centrifuge
  • 37°C incubator
  • Heating block
  • Plastic wrap
  • Autoradiographic film
  • Developer (Kodak GBX)
  • Fixer (Kodak GBX)
  • Thin knife
  • 14‐mL culture tubes
  • Rotomix 50800 shaker (Thermolyne)
  • Lyophilizer
  • Sephadex G‐25 NAP columns, preswollen in H 2O (GE Healthcare)
  • Geiger counter
  • Phosphorimager screen
  • Typhoon 9410 imaging scanner (Amersham Biosciences)
  • ImageQuant software (Molecular Dynamics)
  • Additional reagents and equipment for DNA quantification by UV spectrophotometry (unit 5.2) and polyacrylamide gel electrophoresis (PAGE; appendix 3B)
NOTE: Appropriate precautions must be taken when working with radioactive chemicals to avoid contamination of personnel conducting the study and surroundings. The radioactive waste should be collected and disposed of appropriately following the Nuclear Regulatory Commission (NRC) and institutional guidelines that are provided by the institutional radiation safety officer.NOTE: Use autoclaved deionized water for all reagents.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Böhler, C., Nielsen, P.E., and Orgel, L.E. 1995. Template switching between PNA and RNA oligonucleotides. Nature 376:578‐581.
   Bridson, P.K. and Orgel, L.E. 1980. Catalysis of accurate poly(C)‐directed synthesis of 3′‐5′‐linked oligoguanylates by Zn2+ J. Mol. Biol. 144:567‐577.
   Chaput, J.C. and Switzer, C. 2000a. Nonenzymatic oligomerization on templates containing phosphodiester‐linked acyclic glycerol nucleic acid analogues. J. Mol. Evol. 51:464‐470.
   Chaput, J.C. and Switzer, C. 2000b. Non‐enzymatic transcription of an IsoG⋅IsoC base pair. J. Am. Chem. Soc. 122:12866‐12867.
   Chaput, J.C., Sinha, S., and Switzer, C. 2002. 5‐Propynyluracil⋅diaminopurine: An efficient base‐pair for non‐enzymatic transcription of DNA. Chem. Commun. 15:1568‐1569.
   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.
   Ertem, G. and Ferris, J.P. 1996. Synthesis of RNA oligomers on heterogeneous templates. Nature 379:238‐240.
   Hartel, C. and Göbel, M.W. 2000. Substitution of adenine by purine‐2,6‐diamine improves the nonenzymatic oligomerization of ribonucleotides on templates containing thymidine. Helv. Chim. Acta 83:2541‐2549.
   Heuberger, B.D. and Switzer, C. 2006. Nonenzymatic oligomerization of RNA by TNA templates. Org. Lett. 8:5809‐5811.
   Heuberger, B.D. and Switzer, C. 2008. A pre‐RNA candidate revisited: Both enantiomers of flexible nucleoside triphosphates are DNA polymerase substrates. J. Am. Chem. Soc. 130:412‐413.
   Hill, A.R., Orgel, L.E., and Wu, T. 1993. The limits of template‐directed synthesis with nucleoside‐5′‐phosphoro(2‐methyl)imidazolides. Orig. Life Evol. Biosph. 23:285‐290.
   Huang W. and Ferris, J.P. 2003. Synthesis of 35‐40 mers of RNA oligomers from unblocked monomers. A simple approach to the RNA world. Chem. Comm. 12:1458‐1459.
   Inoue, T. and Orgel, L.E. 1981. Substituent control of the poly (C)‐directed oligomerization of guanosine 5′‐Phosphoroimidazolide. J. Am. Chem. Soc. 103:7666‐7667.
   Inoue, T. and Orgel, L.E. 1982. Oligomerization of (guanosine 5′‐phosphor)‐2‐methylimidazolide on poly(C); An RNA polymerase model. Mol. Biol. J. 162:201‐217.
   Inoue, T. and Orgel, L.E. 1983. Nonenzymatic RNA polymerase model. Science 219:859‐862.
   Johnston, W.K., Unrau, P.J., Lawrence, M.S., Glasner, M.E., and Bartel, D.P. 2001. RNA‐catalyzed RNA polymerization: Accurate and general RNA‐templated primer extension. Science 292:1319‐1325.
   Joyce, G.F., Inoue, T., and Orgel, L.E. 1984a. Non‐enzymatic template‐directed synthesis on RNA random copolymers – Poly(C,U) Templates. J. Mol. Biol. 176:279‐306.
   Joyce, G.F., Visser, G.M., van Boeckel, C.A.A., van Boom, J.H., Orgel, L.E., and van Westrenen, J. 1984b. Chiral selection in poly(C)‐directed synthesis of oligo(G). Nature 310:602‐604.
   Kozlov, I.A. and Orgel, L.E. 1999. Nonenzymatic oligomerization reactions on templates containing inosinic acid or diaminopurine nucleotide residues. Helv. Chim. Acta. 82:1799‐1805.
   Kozlov, I.A., Politis, P.K., Pitsch, S., Herdewijn, P., and Orgel, L.E. 1999. A highly enantio‐selective hexitol nucleic acid template for nonenzymatic oligoguanylate synthesis. J. Am. Chem. Soc. 121:1108‐1109.
   Kozlov, I.A., Zielinski, M., Allart, B., Kerremans, L., Van Aerschot, A., Busso, R., Herdewijn, P., and Orgel, L.E. 2000. Nonenzymatic template‐directed reactions on altritol oligomers, preorganized analogues of oligonucleotides. Chem. Eur. J. 6:151‐155.
   Lohrmann, R. and Orgel, L.E. 1978. Preferential formation of (2′‐5′)‐linked internucleotide bonds in non‐enzymatic reactions. Tetrahedron 34:853‐855.
   Lohrmann, R. and Orgel, L.E. 1980. Efficient catalysis of polycytidylic acid‐directed oligoguanylate formation by Pb2+. J. Mol. Biol. 142:555‐567.
   Mukaiyama, T. and Hashimoto, M. 1971. Phosphorylation of alcohols and phosphates by oxidation‐reduction condensation. Bull. Chem. Soc. Jpn. 44:196‐199.
   Orgel, L.E. 1992. Molecular replication. Nature 358:203‐209.
   Orgel, L.E. 2004. Prebiotic chemistry and the origin of the RNA world. Crit. Rev. Biochem. Mol. Biol. 39:99‐123.
   Orgel, L.E. and Lohrmann, R. 1974. Prebiotic chemistry and nucleic acid replication. Acc. Chem. Res. 7:368‐377.
   Prakash, T.P., Roberts, C., and Switzer, C. 1997. Activity of 2′, 5′‐linked RNA in the template‐directed oligomerization of mononucleotides. Angew. Chem. Int. Ed. Engl. 36:1522‐1523.
   Rothlingshofer, M., Kervio, E., Lommel, T., Plutowski, U., Hochgesand, A., and Richert, C. 2008. Chemical primer extension in seconds. Angew. Chem. Int. Ed. Engl. 47:6065‐6068.
   Rembold, H., Robins, R.K., Seela, K., and Orgel L.E. 1994. Polycytidylate and poly(7‐deazaguanylate): A pair of complementary templates. J. Mol. Evol. 38:211‐214.
   Schöning, K.‐U., Scholtz, P., Guntha, S., Wu, X., Krishnamurthy, R., and Eschenmoser A. 2000. Chemical etiology of nucleic acid structure: the α‐threofuranosyl‐(3′→2′) oligonucleotide system. Science. 290:1347‐1351.
   Schöning, K.‐U., Scholtz, 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.
   Sinha, S., Kim, P.H., and Switzer, C. 2004. 2′,5′‐linked DNA is a template for polymerase‐directed DNA synthesis. J. Am. Chem. Soc. 126:40‐41.
   Sleeper, H.L. and Orgel, L.E. 1979. The catalysis of nucleotide polymerization by compounds of divalent lead. J. Mol. Evol. 12:357‐364.
   Tsai, C.H., Chen, J.Y., and Szostak, J.W. 2007. Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template. Proc. Natl. Acad. Sci. U.S.A. 104:14598‐14603.
   Wu, T. and Orgel, L.E. 1992a. Nonenzymatic template‐directed synthesis on oligodeoxycytidylate sequences in hairpin oligonucleotides. J. Am. Chem. Soc. 114:317‐322.
   Wu, T. and Orgel, L.E. 1992b. Nonenzymatic template‐directed synthesis on hairpin oligonucleotides. 2. Templates containing cytidine and guanosine residues. J. Am. Chem. Soc. 114:5496‐5501.
   Wu, T. and Orgel, L.E. 1992c. Nonenzymatic template‐directed synthesis on hairpin oligonucleotides. 3. Incorporation of adenosine and uridine residues. J. Am. Chem. Soc. 114:7963‐7969.
   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.
   Zhang, L.L., Peritz, A., and Meggers, E. 2005. A simple glycol nucleic acid. J. Am. Chem. Soc. 127:4174‐4175.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library