Preparation of Photoresponsive DNA Tethering Ortho‐Methylated Azobenzene as a Supra‐Photoswitch

Hiroyuki Asanuma1, Hidenori Nishioka1, Teruchika Ishikawa1, Xingguo Liang1

1 Nagoya University, Nagoya, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.45
DOI:  10.1002/0471142700.nc0445s46
Online Posting Date:  September, 2011
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Abstract

This unit describes synthetic procedures of photoresponsive DNA via a phosphoramidite monomer composed of D‐threoninol as a scaffold and 4‐carboxy‐2′,6′‐dimethylazobenzene or 4‐carboxy‐2′‐methylazobenzene that works as a photoswitch more efficiently than previous nonmodified azobenzene (4‐phenylazobenzoic acid). With these newly modified‐azobenzenes, photoregulatory efficiency of DNA hybridization can be greatly improved. Furthermore, thermal stability of cis‐azobenzene of 4‐carboxy‐2′,6′‐dimethylazobenzene remarkably increases compared with the previous non‐modified azobenzene. Curr. Protoc. Nucleic Acid Chem. 46:4.45.1‐4.45.18. © 2011 by John Wiley & Sons, Inc.

Keywords: D‐threoninol azobenzene; phosphoramidite; oligonucleotide; photoregulation; solid‐phase synthesis; thermal stability; hybridization; UV light

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Phosphoramidite Monomer Carrying O‐Methylated Azobenzene on D‐Threoninol
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Photoresponsive DNA Tethering Azobenzene
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Ethyl 4‐nitrobenzoate
  • 2‐Methoxyethanol
  • Ammonium chloride (NH 4Cl)
  • Distilled water
  • Zinc powder
  • Iron(III) chloride hexahydride (FeCl 3⋅6H 2O)
  • Ethanol (EtOH)
  • Ice
  • NaCl
  • Nitrogen source
  • Ice bath
  • Ethyl acetate (AcOEt)
  • Hexane
  • Silica gel (cat. no. 1.07734; 63‐200 µm; Merck)
  • Glacial acetic acid
  • 2‐Methylaniline
  • NaHCO 3
  • Brine (saturated aqueous solution of NaCl)
  • Na 2SO 4
  • 2,6‐Dimethylaniline
  • 2 N NaOH
  • 1 N HCl
  • 100‐, 200‐, and 300‐mL round‐bottomed flasks
  • Magnetic stirrer
  • Teflon‐coated magnetic stirring bar (football‐shaped stirring bar)
  • 50‐ and 500‐mL beakers
  • Condenser
  • Büchner funnels
  • Vacuum filtration system
  • Rotary vacuum evaporator
  • Glass column for the silica‐gel chromatography (diameters of the column are 60 mm and 30 mm)
  • Filter
  • 254‐nm UV lamp
  • Thin‐layer chromatography (TLC) plate (Silica‐gel 60F 254)
  • 200‐mL two‐necked round‐bottomed flasks
  • Flash light
  • pH paper

Basic Protocol 2: Synthesis of Phosphoramidite Monomer Carrying O‐Methylated Azobenzene on D‐Threoninol

  Materials
  • D‐Threoninol (Sigma‐Aldrich, cat. no. 470023)
  • Compound S.3b (see protocol 1)
  • 1‐Hydroxybenzotriazole (HOBt)
  • N,N‐Dimethylformamide (DMF)
  • N,N′‐Dicyclohexylcarbodiimide (DCC)
  • Methanol
  • Chloroform (CHCl 3)
  • Dry pyridine
  • Dry nitrogen
  • Ice bath
  • 4,4′‐Dimethoxytrityl Chloride (DMT‐Cl)
  • Dry dichloromethane (DCM)
  • Ethyl acetate (AcOEt)
  • Triethylamine
  • Hexane
  • 1H‐tetrazole
  • Dry acetonitrile (AN)
  • 2‐Cyanoethyl N, N, N′, N′,‐tetraisopropylphosphordiamidite
  • Sat. aq. NaHCO 3
  • Brine (saturated aqueous solution of NaCl)
  • 50‐, 100‐, and 200‐mL round‐bottomed flasks
  • Teflon‐coated magnetic stirring bar (football‐shaped stirring bar)
  • Magnetic stirrer
  • Thin‐layer chromatography (TLC) plate, silica gel 60 F 254
  • Vacuum filtration system
  • Rotary vacuum evaporator
  • Glass column for the silica‐gel chromatography (diameters of the column are 30 mm)
  • 50‐ and 100‐mL two‐necked round‐bottom flasks
  • Gas balloon
  • 20‐mL two‐necked pear‐shaped flasks
  • Separatory funnels
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D)

Basic Protocol 3: Synthesis, Purification, and Characterization of Photoresponsive DNA Tethering Azobenzene

  Materials
  • Dried compound S.6a or S.6b (see protocol 2)
  • Dry acetonitrile (AN)
  • Conventional phosphoramidite monomers of dA, dG, dC, and dT (Glen Research, cat. nos. 10‐100, 10‐1020, 10‐1010, 10‐1030)
  • CPG support (Glen Research)
  • 28% aqueous ammonia solution (Kishida Reagent Chemicals, cat. no. 000‐03635)
  • 2.0 M triethylamine acetate (TEAA; Glen Research, cat. no. 60‐41110‐60)
  • Deionized water
  • 1.3% aqueous ammonia solution (28% ammonia solution:water = 1:20)
  • Trifluoroacetic acid (TFA; Glen Research, cat. no. 000‐80285)
  • 50% aq. acetonitrile (AN) solution
  • Liquid nitrogen
  • Elution buffer for HPLC purification:
    • Eluent A: aqueous solution of 50 mM ammonium formate
    • Eluent B: acetonitrile/water (1:1 v/v) mixed solution of 25 mM ammonium formate
  • Di‐ammonium hydrogen citrate (as a matrix for MALDI‐TOFMS; Kishida Reagent Chemicals, cat. no. 000‐03782;)
  • 3‐Hydroxypicolinic acid (as a matrix for MALDI‐TOFMS; Tokyo Chemical Industry, cat. no. H0787)
  • 5‐mL Vials
  • DNA synthesizer (e.g., ABI‐3400, Applied Biosystems or H‐8‐SE, Nihon Techno Service)
  • 5‐mL syringes
  • Poly‐Pak cartridge (Glen Research)
  • 2‐mL micro tubes
  • Centrifugal evaporator
  • Reversed‐phase HPLC column [Merck LiChrospher 100 RP‐18(e) column]
  • Additional reagents and equipment for MALDI‐TOFMS (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
   Asanuma, H., Liang, X.G., Nishioka, H., Matsunaga, D., Liu, M.Z., and Komiyama, M. 2007. Synthesis of azobenzene‐tethered DNA for reversible photo‐regulation of DNA. Nat. Protoc. 2:203‐212.
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   Dittmer, W.U. and Simmel, F.C. 2004. Transcriptional control of DNA‐based nanomachines. Nano Lett. 4:689‐691.
   Ito, H., Liang, X.G., Nishioka, H., and Asanuma, H. 2010. Construction of photoresponsive RNA for photoswitching RNA hybridization. Org. Biomol. Chem. 8:5519‐5524.
   Kashida, H., Liang, X.G., and Asanuma, H. 2009. Rational design of functional DNA with a non‐ribose acyclic scaffold. Curr. Org. Chem. 13:1065‐1084.
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   Nishioka, H., Liang, X.G., Kashida, H., and Asanuma, H. 2007. 2′,6′‐Dimethylazobenzene as an efficient and thermo‐stable photo‐regulator for the photoregulation of DNA hybridization. Chem. Commun. 42:4354‐4356.
   Nishioka, H., Liang, X.G., and Asanuma, H. 2010. Effect of the ortho modification of azobenzene on the photoregulatory efficiency of DNA hybridization and thermal stability of its cis‐form. Chem. Eur. J. 16:2054‐2062.
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   Rothemund, P.W.K. 2006. Folding DNA to create nanoscale shapes and patterns Nature 440:297‐302.
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   Zhou, M.G., Liang, X.G., Mochizuki, T., and Asanuma, H. 2010. A light‐driven DNA nanomachine for efficiently photoswitching RNA digestion. Angew. Chem. Int. Ed. 49:2167‐2170.
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