Synthesis of Oligonucleotides Containing 4,5′,8‐Trimethylpsoralen at the 2′‐O Position and Their Cross‐Linking Properties with RNAs

Akio Kobori1, Asako Yamayoshi1, Akira Murakami1

1 Kyoto Institute of Technology, Graduate School of Science and Technology, Department of Biomolecular Engineering, Matsugasaki, Sakyo‐ku, Kyoto
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.15
DOI:  10.1002/0471142700.nc0515s58
Online Posting Date:  September, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

4,5′,8‐Trimethylpsoralen‐conjugated oligonucleotides have been used in the study of photo‐cross‐linking with target oligonucleotides and in the field of the photodynamic therapy. This unit describes synthetic procedures for oligonucleotides using 2′‐O‐methylphosphoramidite units and an adenosine phosphoramidite unit containing a 4,5′,8‐trimethylpsoralen derivative attached at the 2′ position of an adenosine sugar moiety via an ethoxymethylene linkage. Procedures for obtaining the photo‐cross‐linking efficiency of 2′‐O‐methyloligonucleotides containing a 4,5′,8‐trimethylpsoralen derivative with a target oligonucleotide under UV irradiation conditions are also described, together with the procedure for preparation of 32P‐radiolabeled RNA. Curr. Protoc. Nucleic Acid Chem. 58:5.15.1‐5.15.15. © 2014 by John Wiley & Sons, Inc.

Keywords: 4,5′,8‐trimethylpsoralen; antisense; cross‐link

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of N6‐Benzoyl‐5′‐O‐(4,4′‐Dimethyoxytrityl)‐2′‐O‐{[(4,5′,8‐Trimethyl)Psoralen‐4′‐Ylmethoxy]Ethyl}Adenosine‐3′‐O‐(2‐Cyanoethyl)‐N,N‐Diisopropylphosphoramidite 7
  • Basic Protocol 2: Automated Synthesis of 4,5′,8‐Trimethylpsoralen‐Conjugated 2′‐O‐Methyloligoribonucleotides
  • Basic Protocol 3: Evaluation of Photo‐Cross‐Linking Efficiencies of Trimethylpsoralen‐Conjugated 2′‐O‐Methyloligoribonucleotide by Denaturing Polyacrylamide Gel Electrophoresis (Page)
  • 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 N6‐Benzoyl‐5′‐O‐(4,4′‐Dimethyoxytrityl)‐2′‐O‐{[(4,5′,8‐Trimethyl)Psoralen‐4′‐Ylmethoxy]Ethyl}Adenosine‐3′‐O‐(2‐Cyanoethyl)‐N,N‐Diisopropylphosphoramidite 7

  MaterialsNOTE: All reactions should be run under a nitrogen gas atmosphere.
  • 5′‐(2‐Hydroxyethoxymethyl)‐4,5′,8‐trimethylpsoralen (2)
  • Dichloromethane
  • Sodium sulfate (Na 2SO 4)
  • Acetonitrile
  • Pyridine, anhydrous
  • Triphenylphosphine
  • Tetrachloromethane
  • Thin‐layer chromatography (TLC) silica gel plate (TLC Silica gel 60F 254, MERCK)
  • Sodium hydride (NaH), 60% dispersion in mineral oil
  • Adenosine
  • N,N‐Dimethylformamide (DMF)
  • 98:2 (v/v) Dichloromethane/methanol
  • 90:10 (v/v) Chloroform/methanol
  • 50:50 (v/v) Hexane/ethyl acetate
  • Chloroform
  • Saturated aqueous NaHCO 3 solution
  • Wakogel® C‐200 (particle size 75‐150 µm)
  • Sea sand (20‐35 mesh)
  • 5% H 2SO 4 in methanol (TLC dipping reagent)
  • Trimethylchlorosilane
  • Benzoyl chloride
  • 28% Ammonium hydroxide solution
  • 4,4′‐Dimethoxytrityl chloride
  • Hexane
  • Ethyl acetate
  • Diisopropyl ether
  • Bis(N,N,N,N′‐tetraisopropylamino)‐(2‐cyanoethoxy)phosphine
  • 1H‐tetrazole
  • H 2O
  • Magnetic stir bar
  • Rotary evaporator equipped with diaphragm pump
  • 254‐nm UV lamp
  • Glass chromatography column, 6×40 and 6×30 cm
  • Cotton
  • Gel permeation chromatography instrument (JAIGEL‐GS310)
  • Paper filter (Tokyo Roshi; pore size, No.2)
  • 1.5‐mL glass microtube (Maruemu)

Basic Protocol 2: Automated Synthesis of 4,5′,8‐Trimethylpsoralen‐Conjugated 2′‐O‐Methyloligoribonucleotides

  Materials
  • Long‐chain alkylamino controlled‐pore glass (LCAA CPG) supported with 5′‐O‐dimethoxytrityl‐2′‐O‐methyluridine covalently bound through a 3′‐O‐hemisuccinate linker (Glen Research)
  • Acetonitrile (MeCN), anhydrous
  • Standard 2′‐O‐methylribonucleoside phosphoramidites (Glen Research):
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethyoxytrityl)‐2′‐O‐methyladenosine‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethyoxytrityl)‐2′‐O‐methylguanosine‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethyoxytrityl)‐2′‐O‐methylcytidine‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite
    • 5′‐O‐(4,4′‐Dimethyoxytrityl)‐2′‐O‐methyluridine‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite
  • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethyoxytrityl)‐2′‐O‐{[(4,5′,8‐trimethyl)psoralen‐4′‐ylmethoxy]ethyl}  adenosine‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite  (7)
  • Reagents for DNA synthesizer (Glen Research)
  • 0.45 M 5‐benzylthio‐1H‐tetrazol (BMT) in MeCN
  • Tetrahydrofuran (THF)‐acetic anhydride (9:1, v/v; cap A)
  • 10% 1‐Methylimidazole in THF‐pyridine (8:1, v/v; cap B)
  • 0.02 M I 2 in THF‐pyridine‐H 2O (oxidizing solution)
  • 3% Trichloroacetic acid (TCA) in dichloromethane (deblocking solution; Glen Research)
  • 28% Ammonium hydroxide solution
  • Distilled H 2O
  • Acetic acid
  • 1H‐Tetrazole
  • 0.1 M triethylammonium acetate (TEAA) buffer, pH 7.0 (mobile phase A; see recipe)
  • 50% MeCN in 0.1 M triethylammonium acetate (TEAA) buffer, pH 7.0 (mobile phase B; see recipe)
  • Empty synthesis column
  • Glass syringe fitted with Luer lock, needle, and filter (cut from porous polypropylene, 2 mm thick, and forced to base of the barrel)
  • 55°C Heat block (EYELA, MG‐2200)
  • Centrifugal concentrator (TOMY, CC‐105)
  • 2‐mL Screw‐capped tube
  • 1.5‐mL Microtube
  • DNA/RNA synthesizer (Gene World, H‐8 DNA synthesizer)
  • HPLC system with UV detector (SHIMADZU, SCL‐10A, SPD‐M10A, LC‐10ADx2, DGU‐14A)
  • CAPCELL PAK C18 HPLC packed column (SHISEIDO, 5 µm, 4.6 × 150 mm)
  • UV spectrometer (SHIMADZU, UV‐1700)
  • Vortex mixer (Scientific Industries, Vortex‐Genie 2)
  • 1‐mL Plastic syringe
  • 0.45 µm Cellulose acetate filter (ADVANTEC, DISMIC®‐03CP)
  • Lyophilizer (EYELA, FDU‐1000)
  • Additional reagents and equipment for DNA synthesis ( appendix 3A) and HPLC purification (see unit 10.5)

Basic Protocol 3: Evaluation of Photo‐Cross‐Linking Efficiencies of Trimethylpsoralen‐Conjugated 2′‐O‐Methyloligoribonucleotide by Denaturing Polyacrylamide Gel Electrophoresis (Page)

  Materials
  • RNA (FASMAC)
  • 4,5′,8‐Trimethylpsoralen‐conjugated 2′‐O‐methyloligoribonucleotide
  • Adenosine 5′‐triphosphate [γ‐32P]: [γ‐32P] ATP (Perkin Elmer, NEG502Z)
  • T4 polynucleotide kinase with 10× T4 polynucleotide kinase buffer (TAKARA BIO)
  • Sterilized H 2O
  • 1 M aqueous NaCl
  • C18 reversed‐phase spherical silica gel (Wako Pure Chemical Industries, Wakosil®40C18)
  • MeCN
  • 0.1 M triethylammonium acetate (TEAA) buffer, pH 7.0 (see recipe)
  • 50% MeCN in 0.1 M triethylammonium acetate (TEAA) buffer, pH 7.0 (see recipe)
  • M sodium phosphate buffer (pH 7.0; appendix 2A)
  • 7 M Urea
  • N,N,N,N′‐Tetramethylethylenediamine (TEMED)
  • 10% (w/v) Ammonium persulfate (APS)
  • 20% Acrylamide gel stock solution ( appendix 3B)
  • TBE electrophoresis buffer, pH 8.3 ( appendix 2A)
  • Formamide
  • Marker dye (bromophenol blue, xylene cyanol)
  • 0.5‐ and 1.5‐mL Microtube
  • 96‐Well microplate with lid (IWAKI, 3860‐096)
  • 37°C Heat block (EYELA, MG‐2200)
  • 95°C Heat block (EYELA, MG‐2200)
  • Sterilized pipet tip (FCR&Bio, KT‐200Y/Q‐100‐B; Quality Scientific Plastic, 104‐Q)
  • Liquid scintillation counter (PACKARD, A1900CA Tri‐Carb)
  • Lyophilizer (EYELA, FDU‐1000)
  • Transilluminator (FUNAKOSHI FTI‐LW, 365 nm, 1.6 mJ/cm2•s)
  • Gel electrophoresis apparatus
  • IP cassette (FUJIFILM, BAS‐IP‐CST 2340G)
  • BAS‐III imaging plate (FUJIFILM, BAS‐IP MS2325)
  • Imaging analyzer for RI imaging (FUJIFILM, BAS‐2000 BioImaging analyzer)
  • Image Gauge software (FUJIFILM, version 3.4)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Favre, A., Saintome, C., Fourrey, J.L., Clivio, P., and Laugaa, P. 1998. Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid‐protein interactions. J. Photochem. Photobiol. B 42:109‐124.
  Fujimoto, K., Yoshimura, Y., Ikemoto, T., Nakazawa, A., Hayashi, M., and Saito, I. 2005. Photoinduced DNA end capping via N3‐methyl‐5‐cyanovinyl‐2′‐deoxyuridine. Chem. Commun. 25:3177‐3179.
  Geselowitz, D.A. and Neumann, R.D. 1995. Quantitation of triple‐helix formation using a photo‐crosslinkable aryl azide/biotin/oligonucleotide conjugate. Bioconjug. Chem. 6:502‐506.
  Heckman, J.E., Lambert, D., and Burke, J.M. 2005. Photocrosslinking detects a compact, active structure of the hammerhead ribozyme. Biochemistry 44:4148‐4156.
  Higuchi, H., Yamayoshi, A., Yamaguchi, T., Iwase, R., Yamaoka, T., and Murakami, A. 2007. Selective photo‐cross‐linking of 2′‐O‐psoralen‐conjugated oligonucleotide with RNAs having point mutations. Nucleosides Nucleotides Nucleic Acids 26:277‐290.
  Higuchi, M., Yamayoshi, A., Kobori, A., and Murakami, A. 2009. Synthesis of antisense oligonucleotides containing 2′‐O‐psoralenylmethoxyalkyl adenosine for photodynamic regulation of point mutations in RNA. Bioorg. Med. Chem. 17:475‐483.
  Higuchi, M., Yamayoshi, A., Kato, K., Kobori, A., Wake, N., and Murakami, A. 2010. Specific regulation of point‐mutated K‐ras‐immortalized cell proliferation by a photodynamic antisense strategy. Oligonucleotides 20:37‐44.
  Isaacs, S.T., Shen, C‐K.J., Hearst, J.E., and Rapoport, H. 1977. Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA. Biochemistry 16:1058‐1064.
  Kobori, A., Takaya, K., Higuchi, M., Yamayoshi, A., and Murakami, A. 2009. Synthesis and photo‐induced cross‐linking reactions of 4,5′,8‐trimethyl‐psoralen‐incorporated oligodeoxyribonucleotide. Chem. Lett. 38:272‐273.
  Kobori, A., Tomita, K., Nagae, Y., Yamayoshi, A., and Murakami, A. 2012. Synthesis and cross‐linking activity of 4‐N‐(4,5′,8‐trimethylpsoralen‐4‐ylmethyl)‐2′‐deoxycytidine‐containing oligodeoxyribonucleotides. Chem. Lett. 41:804‐805.
  Kolevzon, N. and Yavin, E. 2010. Site‐specific DNA photocleavage and photomodulation by oligonucleotide conjugate. Oligonucleotides 20:263‐275.
  Lee, B.L., Murakami, A., Blake, K.R., Lin, S.B., and Miller, P.S. 1988. Interaction of psoralen‐derivatized oligodeoxyribonucleoside methylphosphonates with single‐stranded DNA. Biochemistry 27:3197‐3203.
  Matsuyama, Y., Yamayoshi, A., Kobori, A., and Murakami, A. 2014. Functional regulation of RNA‐induced silencing complex by photoreactive oligonucleotides. Bioorg. Med. Chem. 22:1003‐1007.
  Murakami, A., Yamayoshi, A., Iwase, R., Nishida, J‐I., Yamaoka, T., and Wake, N. 2001. Photodynamic antisense regulation of human cervical carcinoma cell growth using psoralen‐conjugated oligo(nucleoside phosphorothioate). Eur. J. Pharm. Sci. 13:25‐34.
  Pieles, U. and Englisch, U. 1989. Psoralen covalently linked to oligodeoxyribonucleotides: Synthesis, sequence specific recognition of DNA and photo‐cross‐linking to pyrimidine residues of DNA. Nucleic Acids Res. 17:285‐299.
  Sergiev, P.V., Lavrik, I.N., Wlasoff, V.A., Dokudovskaya, S.S., Dontsova, O.A., Bogdanov, A.A., and Brimacombe, R. 1997. The path of mRNA through the bacterial ribosome: A site‐directed crosslinking study using new photoreactive derivatives of guanosine and uridine. RNA 3:464‐475.
  Takaku, H. and Kamaike, K. 1982. Synthesis of oligoribonucleotides using 4‐methoxybenzyl group as a new protecting group of the 2′‐hydroxyl group of adenosine. Chem. Lett. 11:189‐192.
  Tanaka, T. and Letsinger, R.L. 1982. Syringe method for stepwise chemical synthesis of oligonucleotides. Nucleic Acids Res. 10:3249‐3260.
  Thuong, N.T. and Asseline, U. 1991. Oligonucleotides attached to intercalators, photoreactive and cleavage agents. In Oligonucleotides and Analogues: A Practical Approach. (F. Eckstein, ed.) pp. 283‐306. IRL‐Press, Oxford.
  Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
  Yamaguchi, T. and Saneyoshi, M. 1996. A photolabile 2′, 3′‐dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: Photoaffinity labeling of HIV‐1 reverse transcriptase. Nucleic Acids Res. 24:3364‐3369.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library