Engineering Specific Cross‐Links in Nucleic Acids Using Glycol Linkers

Timothy O'Dea1, Larry W. McLaughlin1

1 Boston College, Chestnut Hill, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.3
DOI:  10.1002/0471142700.nc0503s00
Online Posting Date:  May, 2001
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Abstract

One of the most convenient methods for generating oligonucleotides possessing intra‐ or interstrand cross‐links is through incorporation of oligoethylene glycol bridges by solid‐phase synthesis. The reagents are commercially available or can be synthesized in a few easy synthetic steps. Unlike many other DNA and RNA cross‐links, aspects of the structural and thermodynamic impact of modifying nucleic acids with oligoethylene glycols have been studied. This unit covers protection, phosphitylation, and preparation of the glycol linker for oligonucleotide synthesis.

     
 
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Table of Contents

  • Basic Protocol 1: Protection of the Glycol Chain with a Trityl Group
  • Basic Protocol 2: Phosphitylation of the Monoprotected Glycol Linker
  • Basic Protocol 3: Preparation of Ethylene Glycol Linkers for Incorporation into Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protection of the Glycol Chain with a Trityl Group

  Materials
  • Hexa(ethylene glycol) (HEG)
  • Anhydrous pyridine (preferably freshly distilled)
  • Nitrogen or argon gas
  • 4,4′‐Dimethoxytrityl chloride (DMT‐Cl)
  • 5% (v/v) methanol in dichloromethane
  • 10% (v/v) aqueous sulfuric acid (H 2SO 4; Table A.2A.1)
  • Triethylamine (Et 3N, TEA)
  • Dichloromethane (CH 2Cl 2, DCM; preferably freshly distilled)
  • 5% (w/v) aqueous sodium hydrogen carbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4)
  • Methanol (CH 3OH, MeOH)
  • Non‐acid‐generating desiccant: e.g., sodium hydroxide or calcium carbonate
  • 100‐mL round‐bottom flask and rubber stopper
  • Device for maintaining nitrogen or argon atmosphere (e.g., balloon, syringe, and rubber stopper; see step )
  • Needle and syringe
  • Separatory funnel
  • Silica gel
  • Column for chromatography
  • Rotary evaporator
  • Thin‐layer chromatography (TLC) apparatus (see appendix 3D)
CAUTION: Pyridine and its vapors are toxic; exposure to pyridine must be minimal. The reaction should be performed in a fume hood.

Basic Protocol 2: Phosphitylation of the Monoprotected Glycol Linker

  Materials
  • 4,4′‐Dimethoxytrityl‐protected hexa(ethylene glycol) (DMT‐HEG; see protocol 1)
  • Anhydrous pyridine (preferably freshly distilled; units 3.2)
  • Non‐acid‐generating desiccant: e.g., sodium hydroxide or calcium carbonate
  • Nitrogen or argon gas
  • Anhydrous dichloromethane (CH 2Cl 2, DCM; preferably freshly distilled)
  • Diisopropylethylamine
  • 2‐(Cyanoethyl)‐N,N‐diisopropylchlorophosphoramidite
  • Ethyl acetate
  • 10% (v/v) triethylamine (Et 3N, TEA) in ethyl acetate
  • 5% (w/v) aqueous NaHCO 3
  • Saturated aqueous NaCl
  • Sodium sulfate (Na 2SO 4)
  • 25‐mL round‐bottom flask and rubber stopper
CAUTION: Pyridine and its vapors are toxic; exposure to pyridine must be minimal. The reaction should be performed in a fume hood.

Basic Protocol 3: Preparation of Ethylene Glycol Linkers for Incorporation into Oligonucleotides

  Materials
  • Dimethoxytrityl‐protected hexa(ethylene glycol) phosphoramidite (DMT‐HEG‐P) (see protocol 2)
  • Anhydrous dichloromethane (CH 2Cl 2, DCM; preferably freshly distilled)
  • Anhydrous acetonitrile (preferably freshly distilled)
  • Bottle from DNA synthesizer, tared
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Figures

Videos

Literature Cited

Literature Cited
   Altmann, S., Labhardt, A.M., Bur, D., Lehmann, C., Bannwarth, W., Billeter, M., Wuthrich, K., and Leupin, W. 1995 NMR studies of DNA duplexes singly cross‐linked by different synthetic linkers. Nucl. Acids Res. 23:4827‐4835.
   Amaratunga, M. and Lohman, T.M. 1993. Escherichia coli Rep helicase unwinds DNA by an active mechanism. Biochemistry 32:6815‐6820.
   Benseler, F., Fu, D.J., Ludwig, J., and McLaughlin, L.W. 1993 Hammerhead‐like molecules containing non‐nucleoside linkers are active RNA catalysts. J. Am. Chem. Soc. 115:8483‐8484.
   Cain, R.J. and Glick, G.D. 1998. Use of cross‐links to study the conformational dynamics of triplex DNA. Biochemistry 37:1456‐1464.
   Cload, S.T. and Schepartz, A. 1991. Polyether tethered oligonucleotide probes. J. Am. Chem. Soc. 113:6324‐6326.
   Durand, M., Chevrie, K., Chassignol, M., and Thuong, N.T. 1990. Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain—conformation and stability. Nucl. Acids Res. 18:6353‐6359.
   Ferentz, A.E. and Verdine, G.L. 1991. Disulfide cross‐linked oligonucleotides. J. Am. Chem. Soc. 113:4000‐4002.
   Fu, D.J., Benseler, F., and McLaughlin, L.W. 1994. Hammerhead ribozymes containing non‐nucleoside linkers are active RNA catalysts. J. Am. Chem. Soc. 116:4591‐4598.
   Goodwin, J.T. and Glick, G.D. 1994. Synthesis of a disulfide stabilized RNA hairpin. Tetrahedron Lett. 35:1647‐1650.
   Hendry, P., Moghaddam, M.J., McCall, M.J., Jennings, P.A., Ebel, S., and Brown, T. 1994. Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme. Biochim. Biophys. Acta 1219:405‐412.
   Komatsu, Y., Kanzaki, I., and Ohtsuka, E. 1996. Enhanced folding of hairpin ribozymes with replaced domains. Biochemistry 35:9815‐9820.
   Ma, M.Y.X., McCallum, K., Climie, S.C., Kuperman, R., Lin, W.C., Sumner‐Smith, M., and Barnett, R.W. 1993. Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed, double‐stranded cyclic HIV‐1 TAR RNA analogs with high Tat‐binding affinity. Nucl. Acids Res. 21:2585‐9.
   Moses, A.C., and Schepartz, A. 1996. Triplex tethered oligonucleotide probes. J. Am. Chem. Soc 118:10896‐10897.
   Rajur, S.B., Robles, J., Wiederholt, K., Kuimelis, R.W., and McLaughlin, L.W. 1997. Hoechst 33258 tethered by a hexa(ethylene glycol) linker to the 5′‐termini of oligodeoxynucleotide 15‐mers: Duplex stabilization and fluorescence properties. J. Org. Chem. 62:523‐529.
   Robles, J. and McLaughlin, L.W. 1997. DNA triplex stabilization using a tethered minor‐groove binding Hoechst 33258 analogue. J. Am. Chem. Soc. 119:6014‐6021.
   Robles, J., Rajur, S.B., and McLaughlin, L.W. 1996. A parallel‐stranded DNA triplex tethering a Hoechst 33258 analogue results in complex stabilization by simultaneous major groove and minor groove binding. J. Am. Chem. Soc 118:5820‐5821.
   Thomson, J.B., Tuschl, T., and Eckstein, F. 1993. Activity of hammerhead ribozymes containing non‐nucleotidic linkers. Nucl. Acids Res. 21:5600‐5603.
   Williams, D.J. and Hall, K.B. 1996. Thermodynamic comparison of the salt dependence of natural RNA hairpins and RNA hairpins with non‐nucleotide spacers. Biochemistry 35:14665‐14670.
   Wolfe, S.A. and Verdine, G.L. 1993. Ratcheting torsional stress in duplex DNA. J. Am. Chem. Soc. 115:12585‐12586.
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