Highly Ordered Pyrene π‐Stacks on an RNA Duplex

Mitsunobu Nakamura1, Tadao Takada1, Kazushige Yamana1

1 University of Hyogo, Himeji
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.66
DOI:  10.1002/0471142700.nc0466s63
Online Posting Date:  December, 2015
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Abstract

The syntheses of 2′‐O‐(pyren‐1‐ylmethyl)uridine phosphoramidite, 2′‐O‐(pyren‐1‐ylmethyl)adenosine phosphoramidite, and multiple pyrene‐attached oligo‐RNAs are described in this unit. The 2′‐O‐(pyren‐1‐ylmethyl)nucleosides are converted into the corresponding 2′‐O‐(pyren‐1‐ylmethyl)nucleoside 3′‐phosphoramidites, which can be incorporated into the specific position of oligo‐RNAs by solid‐phase oligonucleotide synthesis. The multiple pyrene‐attached oligo‐RNA forms an A‐form duplex with a complementary multiple pyrene‐attached oligo‐RNA; the pyrenes are associated with π‐stacking along the outside of the duplex. © 2015 by John Wiley & Sons, Inc.

Keywords: phosphoramidite; pyrene; oligoribonucleotides; solid‐phase synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 1‐(Chloromethyl)Pyrene
  • Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(Pyren‐1‐ ylmethyl)Uridine 3′‐[(2‐Cyanoethyl)‐(N,N‐Diisopropyl)] Phosphoramidite
  • Basic Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(Pyren‐1‐Ylmethyl)‐N‐Benzoyladenosine 3′‐[(2‐Cyanoethyl)‐(N,N‐Diisopropyl)]Phosphoramidite
  • Basic Protocol 4: Synthesis and Purification of Oligonucleotides Having Multiple Pyrenes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 1‐(Chloromethyl)Pyrene

  Materials
  • 1‐Pyrenecarboxaldehyde (99% pure, Sigma‐Aldrich)
  • Sodium tetrahydroborate (NaBH 4, purity >95%, Tokyo Kasei)
  • N,N‐Dimethylformamide (DMF, super dehydrated, for organic synthesis, Wako)
  • Thionyl chloride (SOCl 2, purity >98%, Tokyo Kasei)
  • Triethylamine (Et 3N, purity >99%, Nacalai Tesque)
  • Sodium sulfate (Na 2SO 4, anhydrous, purity >98.5%, Nacalai Tesque)
  • Sodium hydrogen carbonate (NaHCO 3, Nacalai Tesque)
  • Phosphorus pentoxide (P 2O 5, purity >98.5%, Tokyo Kasei)
  • Benzene (anhydrous)
  • 35% hydrochloric acid (HCl, Nacalai Tesque)
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH)
  • Argon
  • Three‐necked round‐bottom flasks
  • Magnetic stirrer
  • Stir bar
  • 200‐mL dropping funnel
  • Rubber septum
  • 10‐mL syringe
  • Separatory funnels
  • Rotary evaporator
  • Vacuum oil pump

Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(Pyren‐1‐ ylmethyl)Uridine 3′‐[(2‐Cyanoethyl)‐(N,N‐Diisopropyl)] Phosphoramidite

  Materials
  • Uridine (purity >98%, Tokyo Kasei)
  • 1‐(Chloromethyl)pyrene 1 (prepared in protocol 1)
  • Trityl chloride (TrCl, purity >98%, Tokyo Kasei)
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl, purity >97%, Tokyo Kasei)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphorodiamidite (>94%, Tokyo Kasei)
  • 1H‐Tetrazole
  • Phosphorus pentoxide (P 2O 5, purity >98.5%, Tokyo Kasei)
  • Sodium sulfate (Na 2SO 4, anhydrous, purity >98.5%, Nacalai Tesque)
  • Potassium hydroxide (KOH, purity >85%, Nacalai Tesque)
  • 1,4‐Dioxane (super dehydrated, for organic synthesis, Wako)
  • Tetrahydrofuran (THF, super dehydrated, for organic synthesis, Wako)
  • Pyridine (anhydrous)
  • Benzene (anhydrous)
  • 35% hydrochloric acid (HCl, Nacalai Tesque)
  • Dichloromethane (CH 2Cl 2)
  • Ethyl acetate (AcOEt)
  • Methanol (MeOH)
  • n‐Hexane
  • Acetonitrile (MeCN)
  • Triethylamine (Et 3N)
  • Argon
  • Silica gel (for column chromatography)
  • 5‐mL glass crimp‐top vial and flip off cap
  • Desiccator
  • pH test paper
  • Mortar and pestle
  • Three‐necked round‐bottom flasks
  • Magnetic stirrer
  • Stir bar
  • Rubber septum
  • 5‐ and 10‐mL syringes
  • Separatory funnels
  • Rotary evaporator
  • Vacuum oil pump
  • Chromatography columns (5 × 50 cm, 5 × 30 cm, and 2 × 10 cm)

Basic Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(Pyren‐1‐Ylmethyl)‐N‐Benzoyladenosine 3′‐[(2‐Cyanoethyl)‐(N,N‐Diisopropyl)]Phosphoramidite

  Materials
  • Adenosine (purity >98%, Tokyo Kasei)
  • 1‐(Chloromethyl)pyrene 1 (prepared in protocol 1)
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl, purity >97%, Tokyo Kasei)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphorodiamidite (>94%, Tokyo Kasei)
  • 1H‐Tetrazole
  • Phosphorus pentoxide (P 2O 5, purity >98.5%, Tokyo Kasei)
  • Sodium sulfate (Na 2SO 4, anhydrous, purity >98.5%, Nacalai Tesque)
  • Sodium hydride (NaH)
  • N,N‐Dimethylformamide (DMF)
  • Pyridine (anhydrous)
  • Trimethylsilyl chloride
  • Benzoyl chloride
  • 28% aqueous ammonia (NH 3) solution
  • Dichloromethane (CH 2Cl 2)
  • Ethyl acetate (AcOEt)
  • Triethylamine (Et 3N)
  • Methanol (MeOH)
  • n‐Hexane
  • Acetonitrile (MeCN)
  • Silica gel (for column chromatography)
  • 5‐mL glass crimp‐top vial and flip off cap
  • Desiccator
  • Three‐necked round‐bottom flasks
  • Magnetic stirrer
  • Stir bar
  • Rubber septum
  • 5‐ and 10‐mL syringes
  • 500‐μL microsyringe
  • Separatory funnels
  • Rotary evaporator
  • Vacuum oil pump
  • Chromatography columns (3 × 30 cm, 5 × 50 cm, and 2 × 10 cm)

Basic Protocol 4: Synthesis and Purification of Oligonucleotides Having Multiple Pyrenes

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(pyren‐1‐ylmethyl)uridine 3′‐[(2‐cyanoethyl)‐(N,N‐diisopropyl)]phosphoramidite 3 (prepared in protocol 2)
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(pyren‐1‐ylmethyl)‐N‐benzoyladenosine 3′‐[(2‐cyanoethyl)‐(N,N‐diisopropyl)]phosphoramidite 8 (prepared in protocol 3)
  • RNA phosphoramidites (2′‐OMe‐A‐CE Phosphoramidite, 2′‐OMe‐C‐CE Phosphoramidite, 2′‐OMe‐G‐CE Phosphoramidite, 2′‐OMe‐U‐CE Phosphoramidite, Glen Research)
  • RNA supports (Bz‐A‐RNA‐CPG, 2′‐OMe‐U‐RNA‐CPG, Glen Research)
  • Acetonitrile (MeCN, anhydrous and HPLC grade)
  • 28% aqueous ammonia (NH 3) solution (Nacalai Tesque)
  • Acetic acid
  • Diethyl ether
  • Ammonium formate
  • 1H‐Tetrazole
  • ABI 394 synthesizer
  • Columns for DNA synthesis (Glen Research)
  • Syringes
  • 2‐mL Safe‐Lock centrifuge tubes
  • Dry bath incubator
  • Centrifugal evaporator
  • Lyophilizer
  • Vacuum oil pump
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Figures

Videos

Literature Cited

Literature Cited
  Borer, P.N. 1975. Optical properties of nucleic acids, absorption and circular dichroism spectra. In Handbook of Biochemistry and Molecular Biology, 3rd Edition. Vol. I (G.D. Fasman, ed.), pp. 589‐595. CRC Press, Cleveland.
  Figueira‐Duarte, T.M. and Müllen, K. 2011. Pyrene‐based materials for organic electronics. Chem. Rev. 111:7260‐7314. doi: 10.1021/cr100428a
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  Maie, K., Nakamura, M., Takada, T., and Yamana, K. 2009. Fluorescence quenching properties of multiple pyrene‐modified RNAs. Biorg. Med. Chem. 17:4996‐5000. doi: 10.1016/j.bmc.2009.05.074
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  Nakamura, M., Fukunaga, Y., Sasa, K., Ohtoshi, Y., Kanaori, K., Hayashi, H., Nakano, H., and Yamana, K. 2005a. Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: The structural basis of this difference. Nucleic Acids Res. 33:5887‐5895. doi: 10.1093/nar/gki889
  Nakamura, M., Ohtoshi, Y., and Yamana, K. 2005b. Helical pyrene‐array along the outside of duplex RNA. Chem. Commun. 5163‐5165.
  Nakamura, M., Murakami, Y., Sasa, K., Hayashi, H., and Yamana, K. 2008. Pyrene‐zipper array assembled via RNA duplex formation. J. Am. Chem. Soc. 130:6904‐6905. doi: 10.1021/ja801054t
  Nakamura, M., Okaue, T., Takada, T., and Yamana, K. 2012. DNA‐Templated assembly of naphthalenediimide arrays. Chem. Eur. J. 18:196‐201.doi: 10.1002/chem.201102216
  Nakamura, M., Shimomura, Y., Ohtoshi, Y., Sasa, K., Hayashi, H., Nakano, H., and Yamana, K. 2007. Pyrene aromatic arrays on RNA duplexes as helical templates. Organ. Biomol. Chem. 5:1945‐1951. doi: 10.1039/b705933g
  Okamoto, A., Ichiba, T., and Saito, I. 2004. Pyrene‐labeled oligodeoxynucleotide probe for detecting base insertion by excimer fluorescence emission. J. Am. Chem. Soc. 126:8364‐8365. doi: 10.1021/ja049061d
  Seo, Y.J., Rhee, H., Joo, T., and Kim, B.H. 2007. Self‐duplex formation of an APy‐substituted oligodeoxyadenylate and its unique fluorescence. J. Am. Chem. Soc. 129:5244‐5247. doi: 10.1021/ja069069i
  Wang, T., Sha, R., Dreyfus, R., Leunissen, M.E., Maass, C., Pine, D.J., Chaikin, P.M., and Seeman, N.C. 2012. Self‐replication of information‐bearing nanoscale patterns. Nature 478:225‐228. doi: 10.1038/nature10500
  Wei, B., Dai, M., and Yin, P. 2012. Complex shapes self‐assembled from single‐stranded DNA tiles. Nature 485:623‐626. doi: 10.1038/nature11075
  Wilson, J.N., Teo, Y.N., and Kool, E.T. 2007. Efficient quenching of oligomeric fluorophores on a DNA backbone. J. Am. Chem. Soc. 129:15426‐15427. doi: 10.1021/ja075968a
  Zhu, H. and Lewis, F.D. 2007. Pyrene excimer fluorescence as a probe for parallel G‐quadruplex formation. Bioconjugate Chem. 18:1213‐1217. doi: 10.1021/bc060279u
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