Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling

Ge‐min Fang1, Oliver Seitz1

1 Department of Chemistry, Humboldt‐Universität zu Berlin, Berlin
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.78
DOI:  10.1002/cpnc.44
Online Posting Date:  December, 2017
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Abstract

“Label‐free” fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of “label‐free” bicysteine‐modified PNA probes using solid‐phase synthesis and procedures for sequence‐specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine‐modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence‐specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low‐cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc.

Keywords: fluorescent probes; DNA detection; peptide nucleic acid; single‐nucleobase mutation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of N‐Terminal Bicysteine‐Modified PNA Probes
  • Basic Protocol 2: Synthesis of C‐Terminal Bicysteine‐Modified PNA Probes
  • Basic Protocol 3: Hybridization of PNA Probes 1 and 2, 3 and 4, or 5 and 6 with DNA Templates
  • Basic Protocol 4: Hybridization of PNA Probes 1 and 2 with DNA Template in Cell Lysate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of N‐Terminal Bicysteine‐Modified PNA Probes

  Materials
  • H‐Rink amide chemmatrix resin (35‐ to 100‐mesh particle size, 0.4 to 0.6 mmol/g loading, Sigma‐Aldrich, cat. no. 727768)
  • N,N‐Dimethylformamide (DMF, water content < 150 ppm)
  • Dichloromethane (DCM)
  • Fmoc‐Gly‐OH (Novabiochem)
  • (Benzotriazol‐1‐yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, ≥ 98.5%, Carl Roth)
  • Capping reagent (see recipe)
  • Fmoc‐deprotection reagent (see recipe)
  • N‐Methylmorpholine (NMM, Fisher BioReagents)
  • Fmoc‐PNA‐X(Bhoc)‐OH monomer:
  • Fmoc‐PNA‐A(Bhoc)‐OH (Link Technologies, cat. no. 5001‐C001×1)
  • Fmoc‐PNA‐C(Bhoc)‐OH (Link Technologies, cat. no. 5002‐C001×1)
  • Fmoc‐PNA‐G(Bhoc)‐OH (Link Technologies, cat. no. 5003‐C001×1)
  • Fmoc‐PNA‐T‐OH (Link Technologies, cat. no. 5004‐C001×1)
  • N‐Methyl‐2‐pyrrolidone (NMP, Carl Roth, art. no. P052.1)
  • Fmoc‐Cys(Trt)‐OH (Iris Biotech)
  • 1‐Hydroxybenzotriazole monohydrate (HOBt, Angene Chemical)
  • Resin cleavage reagent (see recipe)
  • Diethylether (Et 2O), ice cold
  • Acetonitrile (HPLC‐super gradient ACN reagent, water content < 30 ppm)
  • HPLC eluent A (see recipe)
  • HPLC eluent B (see recipe)
  • Frit‐fitted 5‐mL plastic syringes
  • Shakers, room temperature and 37ºC
  • 15‐mL plastic tubes
  • Vacuum
  • 2‐mL plastic vials
  • Centrifuge (ThermoFisher Scientific)
  • 1‐cm quartz cuvette
  • Bio‐RAD smartspecTM plus spectrophotometer
  • Microscale column (1‐ to 5‐µmol, Intavis AG Bioanalytic Instruments)
  • High‐performance liquid chromatography (HPLC) system (Agilent 1100 series)
  • Preparative column: VP 250/21 Nucleodur C18, gravity 5 µm (Macherey Nagel, cat. no. 762102)
  • Analytical column: cat. ZP 250110 Nucleodur C18, gravity 5 µm (Macherey Nagel, cat. no. 762113400)
  • UPLC‐MASS (ESI‐MS with a VL quadrupole mass spectrometer, Waters Corporation)
  • Lyophilizer (Zirbus Technology)

Basic Protocol 2: Synthesis of C‐Terminal Bicysteine‐Modified PNA Probes

  Additional Materials (also see protocol 1)
  • DNA samples (dH, dH mut1, dH mut2, rasT, and rasG, each 50‐nmol scale) (DNA oligonucleotides ordered from BioTez GmBH or Biomers GmbH)
  • RNase‐free water
  • PNA probes
  • FIAsH‐EDT 2 (Santa Cruz Biotech, cat. no. Sc‐363644)
  • ReAsH‐EDT 2 (Santa Cruz Biotech, cat. no. Sc‐391916)
  • CrAsH‐EDT 2 (prepared as reported by Cao et al., 2006)
  • Dimethyl sulfoxide (DMSO)
  • Sodium chloride (NaCl)
  • Sodium dihydrogen phosphate (NaH 2PO 4·2H 2O)
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP·HCl)
  • 1 M sodium hydroxide (NaOH)
  • 1,2‐Ethanethiol (EDT, Sigma‐Aldrich, cat. no. 02390)
  • NanoDrop ND‐1000 spectrophotometer (peQlab Biotechnologie)
  • 1.0‐mL microcentrifuge tubes
  • 1.0‐mL quartz cuvettes
  • Cary Eclipse fluorescence spectrophotometer
  • Cary 100 UV‐visible spectrophotomer

Basic Protocol 3: Hybridization of PNA Probes 1 and 2, 3 and 4, or 5 and 6 with DNA Templates

  Additional Materials (also see Basic Protocols protocol 11, protocol 22, and protocol 33)
  • DMEM medium (containing 1% penicillin‐streptomycin and 10% fetal calf serum)
  • HEK293 cell line
  • Refrigerated centrifuge
  • French Press Cell Disruptor (Thermo Electron Corporation)
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Figures

Videos

Literature Cited

Literature Cited
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  Gorska, K. & Winssinger, N. (2013). Reactions templated by nucleic acids: More ways to translate oligonucleotide‐based instructions into emerging function. Angewante Chemie International Edition, 52, 6820–6843. doi: 10.1002/anie.201208460.
  Griffin, B. A., Adams, S. R., & Tsien, R. Y. (1998). Specific covalent labeling of recombinant protein molecules inside live cells. Science, 281, 269–272. doi: 10.1126/science.281.5374.269.
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  Hövelmann, F., Gaspar, I., Loibl, S., Ermilov, E. A., Röder, B., Wengel, J., … Seitz, O. (2014). Brightness through local constraint‐LNA‐enhanced FIT hybridization probes for in vivo ribonucleotide particle tracking. Angewante Chemie International Edition, 53, 11370–11375. doi: 10.1002/anie.201406022.
  Hövelmann, F. & Seitz, O. (2016). DNA stains as surrogate nucleobases in fluorogenic hybridization probes. Accounts of Chemical Research, 49, 714–723. doi: 10.1021/acs.accounts.5b00546.
  Luedtke, N. W., Dexter, R. J., Fried, D. B., & Schepartz, A. (2007). Surveying polypeptide and protein domain conformation and association with FIAsH and ReAsH. Nature Chemical Biology, 3, 779–784. doi: 10.1038/nchembio.2007.49.
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  Takei, F., Suda, H., Hagihara, M., Zhang, J., Kobori, A., & Nakatani, K. (2007). Allele specific C‐bulge probes with one unique fluorescent molecule discriminate the single nucleotide polymorphism in DNA. Chemistry‐A European Journal, 13, 4452–4457. doi: 10.1002/chem.200601496.
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