Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs

John J. Turner1, Donna Williams1, David Owen1, Michael J. Gait1

1 Medical Research Council, Laboratory of Molecular Biology, Cambridge
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.28
DOI:  10.1002/0471142700.nc0428s24
Online Posting Date:  April, 2006
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Abstract

Peptide conjugation of oligonucleotides and their analogs is being studied widely towards improving the delivery of oligonucleotides into cells. Amongst the many possible routes of conjugation, the disulfide linkage has proved to be the most popular. This reversible linkage may have advantages for cell delivery, since it is likely to be cleaved within cells, thus releasing the oligonucleotide cargo. It is straightforward to introduce thiol functionalities into both oligonucleotide and peptide components suitable for disulfide conjugation. However, severe difficulties have been encountered in carrying out conjugations between highly cationic peptides and negatively charged oligonucleotides because of aggregation and precipitation. Presented here are reliable protocols for disulfide conjugation that have been verified for both cationic and hydrophobic peptides as well as oligonucleotides containing deoxyribonucleosides, ribonucleosides, 2′‐O‐methylribonucleosides, locked nucleic acid (LNA) units, as well as phosphorothioate backbones. Also presented are reliable protocols for disulfide conjugation of peptide nucleic acids (PNAs) with peptides.

Keywords: conjugation; disulfide oligonucleotide; peptide; PNA

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Conjugation of Peptides with Oligonucleotide Analogs Containing Negatively Charged Phosphates
  • Alternate Protocol 1: Conjugation of C‐Terminal Cys‐Containing Peptides to Oligonucleotides via Activation of the Oligonucleotide
  • Basic Protocol 2: Conjugation of Peptides with Peptide Nucleic Acids
  • Support Protocol 1: Determination of Molecular Mass by MALDI‐TOF Mass Spectrometry
  • Support Protocol 2: Determination of Thiol Content by the Ellman's Test
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Conjugation of Peptides with Oligonucleotide Analogs Containing Negatively Charged Phosphates

  Materials
  • Fmoc‐protected amino acid monomers (Novabiochem) including:
    • Fmoc‐Arg(Pbf)‐OH
    • Fmoc‐Asn(Tr)‐OH
    • Fmoc‐Cys(Tr)‐OH
    • Fmoc‐Gln(Tr)‐OH
    • Fmoc‐Glu(OtBu)‐OH
    • Fmoc‐His(Tr)‐OH
    • Fmoc‐Lys(Boc)‐OH
    • Fmoc‐Trp(Boc)‐OH
  • N,N‐Dimethylformamide (DMF, AnalaR‐grade, BDH Chemicals), freshly distilled
  • PyBop (Novabiochem)
  • N,N‐Diisopropylethylamine (DIPEA, 99+%, Applied Biosystems)
  • Piperidine (>99.5%, Romil)
  • NovaSyn TGR resin (for C‐terminal amide synthesis, Novabiochem)
  • PEG‐PS resin (for C‐terminal carboxylic acid synthesis, Applied Biosystems)
  • Boc‐Cys(Npys)‐OH (for N‐terminal cysteine; Bachem Bioscience)
  • Isopropanol
  • Trifluoroacetic acid (TFA, >99.9%, Romil)
  • Triisopropylsilane (TIS, >99%, Aldrich)
  • Diethyl ether, 4°C
  • Acetonitrile (MeCN, HPLC‐grade, Fisher Scientific)
  • Millipore water or double‐distilled deionized water
  • 1,2‐Ethanedithiol (EDT, >98%, Fluka)
  • Water (HPLC‐grade)
  • 1.0 M NH 4HCO 3 solution (aq.)
  • 10 mg/mL 2‐aldrithiol (Aldrich) in DMF
  • Nucleoside phosphoramidites (as needed):
    • 2′‐Deoxyribonucleoside phosphoramidites (Glen Research)
    • 2′‐O‐Me‐ribonucleoside phosphoramidites (Transgenomics)
    • Locked nucleic acid (LNA) phosphoramidites (Link Technologies)
  • Anhydrous acetonitrile
  • 3′‐(6‐Fluorescein)‐CPG (for 3′‐fluorescent oligonucleotides, Glen Research)
  • Thiol modifier C6‐S‐S (for 5′‐thiol modification, Glen Research)
  • 0.02 M iodine solution in 78:2:20 (v/v/v) THF/pyridine/water (Proligo)
  • 5‐Ethylthio‐1H‐tetrazole (a 0.25 M solution in MeCN, Link Technologies)
  • 30% (v/v) aq. ammonia
  • Sodium perchlorate (AnalaR, BDH Chemicals)
  • 2.0 M Tris·Cl, pH 6.8
  • Formamide (p.a. ≥99.0%, Fluka)
  • Sterilized water
  • 2.0 M triethylammonium acetate, pH 7 (TEAA, Glen Research)
  • 1.0 M aqueous D/L‐dithiothreitol (DTT, ≥99%, Aldrich)
  • Triethylamine (TEA, ≥99.5%, Fluka)
  • Peptide synthesizer (e.g., APEX 396 or Pioneer peptide synthesizer)
  • Desiccator attached to vacuum
  • 15‐mL polyethylene syringe (IST empty reservoir, Kinesis)
  • 20‐µm polyethylene frit (Kinesis)
  • Speedvac concentrator
  • Benchtop centrifuge
  • 15‐ and 50‐mL centrifuge tubes (Falcon)
  • 0.22‐ (water) and 0.45‐µm (water and organic) filters (Millipore)
  • HPLC system, chemically inert, suitable for ion‐exchange chromatography, with:
    • PEEK tubing throughout
    • Injector, sample loop, and syringe (manual loading)
    • UV/Vis detector, variable wavelength 190 and 500 nm (preferable) or dual‐wavelength detection
    • Helium for degassing
    • Jupiter reversed‐phase HPLC column with guard (analytical and semi‐prep, Phenomenex)
    • DNAPac PA‐100 (9 × 250–mm) Dionex column (semi‐prep) with guard attached
    • Resource Q column (1 mL/min analytical or 6 mL/min semi‐prep, Amersham Biosciences)
  • Lyophilizer
  • Rotary evaporator and water pump
  • 1.5‐mL screw‐cap tube (Sarstedt) or vial
  • 55°C bath or temperature block (optional)
  • Spin‐X tube (Costar)
  • Dialysis tubing, 3500 MWCO (Medicell International Ltd.)
  • UV spectrometer
  • Sephadex NAP‐25 column (Amersham Biosciences)
  • 0.5‐mL microcentrifuge tube
  • Slide‐a‐lyzer (0.5‐ to 3‐mL capacity, 3500 MWCO, Pierce)
  • Additional reagents and equipment for peptide synthesis, MALDI‐TOF‐MS (see protocol 4), Ellman's test (see protocol 5), oligonucleotide synthesis ( appendix 3C), and amino acid analysis (optional; see unit 4.22)
NOTE: Amino acid protecting groups are tBu, tert‐butyl; Boc, tert‐butyloxycarbonyl; Fmoc, 9‐fluorenylmethoxycarbonyl; Pbf, 2,2,4,6,7‐pentamethyldihydrobenzofuran‐5‐sulfonyl; pys, pyridylsulfenyl; Npys, 3‐nitropyridylsulfenyl; and Tr, trityl.

Alternate Protocol 1: Conjugation of C‐Terminal Cys‐Containing Peptides to Oligonucleotides via Activation of the Oligonucleotide

  • Sephadex NAP‐10 column (Amersham Biosciences)

Basic Protocol 2: Conjugation of Peptides with Peptide Nucleic Acids

  Materials
  • Fmoc (Bhoc) PNA monomers (Applied Biosystems)
  • N‐Methylpyrrolidinone (NMP, ≥99.5%, Fluka)
  • PyBop (Novabiochem)
  • N,N‐Dimethylformamide (DMF, AnalaR‐grade, BDH Chemicals), freshly distilled
  • N,N‐Diisopropylethylamine (DIPEA, 99+%, Applied Biosystems)
  • 2,6‐Lutidine (≥99%, Aldrich)
  • Piperidine (>99.5%, Romil)
  • Fmoc‐PAL‐PEG‐PS amide resin (Applied Biosystems)
  • Isopropanol
  • Trifluoroacetic acid (TFA, >99.9%, Romil)
  • Triisopropylsilane (TIS, >99%, Aldrich)
  • Phenol
  • Diethyl ether, 4°C
  • 5% acetic anhydride/6% 2,6‐lutidine solution in DMF (PNA capping solution, Applied Biosystems)
  • 1.0 M aq. NH 4OAc (AnalaR‐grade, BDH Chemicals)
  • APEX 396 Robotic peptide synthesizer
  • 1‐mL polyethylene syringe (IST empty reservoir, Kinesis)
  • 10‐µm polyethylene frit (Kinesis)
  • Plastic tap
  • Filtration unit
  • 15‐mL centrifuge tube (Falcon)
  • Heating jacket for HPLC column
  • UV spectrometer
  • Lyophilizer
  • Additional reagents and equipment for reversed‐phase HPLC (see protocol 1)

Support Protocol 1: Determination of Molecular Mass by MALDI‐TOF Mass Spectrometry

  Materials
  • α‐Cyano‐4‐hydroxycinnamic acid (CHCA, ≥99.0%, Aldrich)
  • Acetonitrile
  • 3% (v/v) aq. trifluoroacetic acid (TFA, >99.9%, Romil)
  • 2,6‐Dihydroxyacetophenone (DHAP, ≥99.0%, Fluka)
  • Methanol
  • Diammonium hydrogen citrate (≥99.0%, Fluka)
  • Millipore water or double‐distilled deionized water
  • 2,4,6‐Trihydroxyacetophenone (THAP, ≥99%, Fluka)
  • 1.5‐mL solvent‐resistant microcentrifuge tubes
  • MALDI‐TOF mass spectrometer

Support Protocol 2: Determination of Thiol Content by the Ellman's Test

  Materials
  • Ellman's reagent: 2 mM dithio‐bis‐2‐nitrobenzoic acid (DTNB) in 50 mM sodium acetate (NaOAc)
  • 2.0 M Tris·Cl, pH 8.0
  • UV spectrometer with 1‐mL, 1‐cm path length quartz glass cuvette (Suprasil, Hellma)
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Figures

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Literature Cited

Literature Cited
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