Synthesis of Fluorescent Potassium Ion–Sensing Probes Based on a Thrombin‐Binding DNA Aptamer–Peptide Conjugate

Shigeori Takenaka1

1 Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.9
DOI:  10.1002/0471142700.nc0809s62
Online Posting Date:  September, 2015
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Abstract

This unit provides a procedure for synthesis of the potassium‐sensing peptide‐oligodeoxyribonucleotide conjugate PSO‐5 for visualizing potassium ions (K+) in living cells. It is constructed by combining an oligodeoxyribonucleotide carrying a thrombin‐binding DNA aptamer (TBA) sequence with an uncharged peptide carrying biotin and the fluorescence tags fluorescein (FAM) and tetramethylrhodamine (TAMRA). The PSO‐5 and biotin‐modified nuclear export signal peptide are conjugated through streptavidin, and this sensing molecule is introduced into the cell where it is localized in the cytoplasm. The TBA part of PSO‐5 shows a conformational change from a random coil to a tetraplex structure induced by K+ and a change in the fluorescence resonance energy transfer (FRET) efficiency between FAM and TAMRA arising from its conformational change, enabling fluorometric detection of changes in K+ concentration. © 2015 by John Wiley & Sons, Inc.

Keywords: potassium ion–sensing; thrombin‐binding DNA aptamer (TBA); fluorescence resonance energy transfer (FRET)

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Fmoc amino acids and resin (see Table 8.9.1 for solutions)
    • Fmoc‐NH‐SAL resin (Watanabe Chemical Industries)
    • Fmoc‐Gly‐OH (Watanabe Chemical Industries)
    • Fmoc‐Cys(Trt)‐OH (Watanabe Chemical Industries)
    • Fmoc‐Lys(Boc)‐OH (Watanabe Chemical Industries)
    • Fmoc‐Acp(6)‐OH (6‐[(9‐fluorenylmethoxycarbonyl)amino]hexanoic acid; Watanabe Chemical Industries)
  • 30% (v/v) piperidine (Aldrich) in DMF (see Table 8.9.1 for volumes)
  • Fmoc‐NH‐SAL resin (Watanabe Chemical Industries; Table 8.9.1)
  • N,N‐Dimethylformamide (DMF), SP grade (Watanabe Chemical Industries): dehydrate further by holding for 1 day after adding 0.5‐nm molecular sieves (2‐mm bead diameter; Merck Millipore), according to the manufacturer's directions
  • Coupling agents R1 and R2 (Table 8.9.1)
    • 1‐[Bis(dimethylamino)methyliumyl]‐1 H‐benzotriazole‐3‐oxide hexafluorophosphate, anhydrous (HBTU; Watanabe Chemical Industries; see Table 8.9.1 for solution)
    • 1‐Hydroxy‐1 H‐benzotriazole, anhydrous (HOBT; Watanabe Chemical Industries; see Table 8.9.1 for solution)
    • Ethyldiisopropylamine (DIEA; Tokyo Chemical company; see Table 8.9.1 for solution)
  • Biotin (Aldrich; see Table 8.9.1 for solution)
  • Triethylamine, anhydrous (TEA; Wako Pure Chemical Industries): dehydrate further by holding for 1 day after adding 0.5‐nm molecular sieves (2‐mm bead diameter; Merck Millipore), according to the manufacturer's directions
  • Dimethyl sulfoxide (DMSO), dehydrated (Wako Pure Chemical Industries): dehydrate further by holding for 1 day after adding 0.5‐nm molecular sieves (2‐mm bead diameter; Merck Millipore), according to the manufacturer's directions
  • Cleavage cocktail (see recipe)
  • Ethyl ether (Wako Pure Chemical Industries)
  • Ethyl acetate (Wako Pure Chemical Industries)
  • Eluent A: 0.1% (v/v) trifluoroacetic acid (TFA; Wako Pure Chemical Industries) in Milli‐Q water (Millipore)
  • Eluent B: 70% (v/v) acetonitrile in 0.1 % (v/v) TFA in Milli‐Q water
  • 53.1 nmol oligodeoxyribonucleotide 1 carrying a TBA sequence (5′‐GGT TGG TGT GGT TGG TT‐3′) with an amino moiety and FAM at its 5′‐ and 3′‐termini, respectively (custom synthesis; Sigma‐Aldrich)
  • 3‐Hydroxy propionic acid
  • PBS (phosphate‐buffered saline; appendix 2A)
  • Sulfosuccinimidyl 4‐[N‐maleimidomethyl]cyclohexane‐1‐carboxylate (sulfo‐SMCC; Thermo Scientific)
  • Milli‐Q water (Millipore)
  • 1 M potassium phosphate buffer ( appendix 2A)
  • Buffer A: 10% (v/v) acetonitrile/0.1 M TEAA buffer, pH 7.0 ( appendix 2A)
  • Buffer B: 10% (v/v) acetonitrile/0.1 M TEAA buffer, pH 7.0 ( appendix 2A; dilute from 1 M)
  • 5‐Carboxytetramethylrhodamine NHS ester, single isomer (5‐TAMRA‐SE; emp Biotech GmbH)
  • 30% (w/v) of (19:1) acrylamide/bis acrylamide (Nakalai Tesque)
  • 10‐bp DNA ladder (Promega)
  • 6× loading buffer (TaKaRa): 36% glycerol/30 mM EDTA/0.05% bromophenol blue (BPB)/0.035% xylene cyanol (XC)
  • 10× TBE buffer ( appendix 2A): dilute as required before use
  • 10,000× GelStar nucleic acid stain in DMSO (TaKaRa Bio)
  • Peptide synthesizer (PSSM‐8, Shimadzu; http://pdf.medicalexpo.com/pdf/shimadzu/pssm‐8‐system/71044‐102231.html) with syringe‐type reaction vessels and vials, and bottles for coupling agents)
  • 0.45‐μm Millex‐HV filter, PVDF (33 mm, nonsterile (Merck Millipore)
  • 2.1 × 150–mm Inertsil ODS‐3 column (2‐μm particle size; GL Sciences)
  • 1.5‐mL clear, round‐bottom microcentrifuge tubes (e.g., Hyper microtubes; Watson Bio Lab)
  • Vacuum freeze‐dryer FD‐1000 (EYELA, http://www.eyelaworld.com/product_view.php?id=126)
  • Intelli‐Mixer RM‐2 M (ELMI, http://www.elmi‐tech.com/rm)
  • illustra NAP‐5 HPLC columns (up to 0.5‐mL sample volume; GE Healthcare)
  • 4.6 × 250–mm Mightysil RP‐18 column (3‐μm particle size; Kanto Chemical)
  • illustra NAP‐10 HPLC columns (up to 1.0‐mL sample volume; GE Healthcare)
  • Slab type gel electrophoresis apparatus (e.g., AE‐6530; ATTO).
  • Additional reagents and equipment for carrying out high‐performance liquid chromatography (HPLC; unit 10.5; Sinha et al., 2015), MALDI‐TOF mass spectrometry (unit 4.28; Turner et al., ), and nondenaturing polyacrylamide gel electrophoresis (PAGE; Gallagher, )
Table 8.9.1   MaterialsReagents Used for Peptide Synthesis

Reagent Amount Mole (μmol) Solvent Comment
Fmoc‐NH‐SAL resin 30 mg 1.65 0.55 mmol/g
Fmoc‐Gly‐OH 49 mg 16.5 198 μL DMF 10‐fold molar excess
Fmoc‐Cys(Trt)‐OH 97 mg 16.5 198 μL DMF 10‐fold molar excess
Fmoc‐Lys(Boc)‐OH 77 mg 16.5 198 μL DMF 10‐fold molar excess
Fmoc‐Acp(6)‐OH 58 mg 16.5 198 μL DMF 10‐fold molar excess
Biotin 40 mg 6.6 20 μL TEA 178 μL DMSO 4‐fold molar excess
R1
1‐[Bis(dimethylamino)methyliumyl]‐1 H‐benzotriazole‐3‐oxide hexafluorophosphate (HBTU) 1.8 g 9.6 mL DMF Coupling reagent
1‐Hydroxy‐1 H‐benzotriazole (HOBT) 0.74 g
R2
N‐Ethyldiisopropylamine (DIEA) 1.68 mL 7.9 mL DMF Coupling reagent
Other solutions
30% (w/v) piperidine 8.7 mL 20.3 mL DMF Decoupling reagent
N,N‐Dimethylformamide (DMF) 3 L Washing solvent

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Figures

Videos

Literature Cited

Literature Cited
  Gallagher, S.R. 1999. One‐dimensional electrophoresis using nondenaturing conditions. Curr. Protoc. Mol. Biol. 47:10.2B.1‐10.2B.11.
  Goodchild, A., King, A., Gozar, M.M., Passioura, T., Tucker, C., and Rivory, L., 2007. Cytotoxic G‐rich oligodeoxynucleotides: Putative protein targets and required sequence motif. Nucleic. Acids Res. 35:4562‐4572. doi: 10.1093/nar/gkm465.
  Harrison, J.G., and Balasubramanian, S. 1998. Synthesis and hybridization analysis of a small library of peptide‐oligonucleotide conjugates. Nucleic Acids Res. 26:3136‐3145. doi: 10.1093/nar/26.13.3136.
  Juskowiak, B. and Takenaka, S. 2006. Fluorescence energy transfers in the studies of guanine quadruplexes. In Methods in Molecular Biology, Vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes. Designs and Protocols (Vladimir V. Didenko, M.D. Ph.D.) pp. 311‐341. Humana Press, Totowa, NJ.
  Lewenstam, A., Maj‐Zurawska, M., and Hulanicki, A. 1991. Application of ion‐selective electrodes in clinical analysis. Electroanalysis 3:727‐734. doi: 10.1002/elan.1140030802.
  Meuwis, K., Boens, N., De Schryver, F.C., Gallay, J., and Vincent, M. 1995. Photophysics of the fluorescent K+ indicator PBFI. Biophys. J. 68:2469. doi: 10.1016/S0006‐3495(95)80428‐5.
  Nagatoishi, S., Nojima, T., Juskowiak, B., and Takenaka, S. 2005. Pyrene‐labeled G‐quadruplex oligonucleotide as a fluorescence probe for potassium ion detection in biological applications. Angew. Chem. Int. Ed. 44:5067‐5070. doi: 10.1002/anie.200501506.
  Nagatoishi, S., Nojima, T., Galezowska, E., Juskowiak, B., and Takenaka, S. 2006. G‐quadruplex‐based FRET probes with the thrombin‐binding aptamer (TBA) sequence designed for the efficient fluorometric detection of potassium ion. Chem. Bio. Chem. 7:1730‐1737. doi: 10.1002/cbic.200600179.
  Ohtsuka, K., Sato, S., Sato, Y., Sota, K., Ohzawa, S., Matsuda, T., Takemoto, K., Takamune, N., Juskowiak, B., Nagai, T., and Takenaka, S. 2012. Fluorescence imaging of potassium ions in living cells using a fluorescent probe based on a thrombin binding aptamer‐peptide conjugate. Chem. Comm. 48:4740‐4742. doi: 10.1039/c2cc30536d.
  Sinha, N.D. and Jung, K.E. 2015. Analysis and purification of synthetic nucleic acids using HPLC. Curr. Protoc. Nucleic Acid Chem. 61:10.5.1‐10.5.39. doi: 10.1002/0471142700.nc1005s61.
  Takenaka, S. and Juskowiak, B. 2011. Fluorescence detection of potassium ion using G‐quadruplex structure. Anal. Sci. 27:1167‐1172. doi: 10.2116/analsci.27.1167.
  Turner, J.J., Williams, D., Owen, D., and Gai, M.J. 2006. Disulfide conjugation of peptides to oligonucleotides and their analogs. Curr. Protoc. Nucleic Acid Chem. 24:4.28.1‐4.28.21.
  Wen, W., Meinkoth, J.L., Tsien, R.Y., and Taylor, S.S. 1995. Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463‐473. doi: 10.1016/0092‐8674(95)90435‐2.
  Yu, S.P., Canzoniero, M.T., and Choi, D.W. 2001. Ion homeostasis and apoptosis. Curr. Opin. Cell Biol. 13:405‐411. doi: 10.1016/S0955‐0674(00)00228‐3.
Key Reference
  Juskowiak, B. and Takenaka, S. 2006. Fluorescence energy transfers in the studies of guanine quadruplexes. In Methods in Molecular Biology, Vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes. Designs and Protocols (Vladimir V. Didenko, M.D. Ph.D.) pp. 311‐341. Humana Press, Totowa, NJ.
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