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


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


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

  • 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

  • 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

  • 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

  • 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)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Antopolsky, M., Azhayeva, E., Tengvall, U., Auriola, S., Jääskeläinen, I., Rönkkö, S., Honkakoski, P., Urtti, A., Lönnberg, H., and Azhayev, A. 1999. Peptide‐oligonucleotide phosphorothioate conjugates with membrane translocation and nuclear localization properties. Bioconjug. Chem. 10:598‐606.
   Astriab‐Fisher, A., Sergueev, D.S., Fisher, M., Shaw, B.R., and Juliano, R.L. 2000. Antisense inhibition of P‐glycoprotein expression using peptide‐oligonucleotide conjugates. Biochem. Pharmacol. 60:83‐90.
   Bennett, C.F., Chiang, M.‐Y., Chan, H., Shoemaker, J.E.E., and Mirabelli, C.K. 1992. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41:1023‐1033.
   Bongartz, J.P., Aubertin, A.M., Milhaud, P.G., and Lebleu, B. 1994. Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide. Nucl. Acids Res. 22:4681‐4688.
   Braun, K., Peschke, P., Pipkorn, R., Lampel, S., Wachsmuth, M., Waldeck, W., Friedrich, E., and Debus, J. 2002. A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells. J. Mol. Biol. 318:237‐243.
   Chiu, Y.‐L., Ali, A., Chu, C., Cao, H., and Rana, T.M. 2004. Visualizing a correlation between siRNA, localization, cellular uptake and RNAi in living cells. Chem. Biol. 11:1165‐1175.
   Corey, C.R. 1995. 48000‐fold acceleration of hybridisation by chemically modified oligonucleotides. J. Amer. Chem. Soc. 117:9373‐9374.
   Eritja, R., Pons, A., Escarceller, M., Giralt, E., and Albericio, F. 1991. Synthesis of defined peptide‐oligonucleotide hybrids containing a nuclear transport signal sequence. Tetrahedron 47:4113‐4120.
   Gait, M.J. 2003. Peptide‐mediated cellular delivery of antisense oligonucleotides and their analogues. Cell. Mol. Life Sci. 60:1‐10.
   Juliano, R.L. 2005. Peptide‐oligonucleotide conjugates for the delivery of antisense and siRNA. Curr. Opin. Mol. Ther. 7:132‐138.
   Kaplan, I.M., Wadia, J.S., and Dowdy, S.F. 2005. Cationic TAT peptide transduction domain enters cells by macropinocytosis. J. Control Release 102:247‐253.
   Kaushik, N., Basu, A., Palumbo, P., Nyers, R.L., and Pandey, V.N. 2002. Anti‐TAR polyamide nucleotide analog conjugated with a membrane‐permeating peptide inhibits Human Immunodeficiency Virus Type I production. J. Virol. 76:3881‐3891.
   Kilk, K., Elmquist, A., Saar, K., Pooga, M., Land, T., Bartfai, T., Soomets, U., and Langel, U. 2004. Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA. Neuropeptides 38:316‐324.
   Koppelhus, U., Awasthi, S.K., Zachar, V., Holst, H.U., Ebbeson, P., and Nielsen, P.E. 2002. Cell‐dependent differential cellular uptake of PNA, peptides and PNA‐peptide conjugates. Antisense & Nucleic Acid Drug Dev. 12:51‐63.
   Lindgren, M., Hällbrink, M., Prochiantz, A., and Langel, U. 2000. Cell‐penetrating peptides. Trends Pharmacol. Sci. 21:99‐103.
   Lindsay, M.A. 2002. Peptide‐mediated cell delivery: Application in protein target validation. Curr Opin Pharmacol 2:587‐594.
   Lochmann, D., Jauk, E., and Zimmer, A. 2004. Drug delivery of oligonucleotides by peptides. Eur. J Pharm. Biopharm. 58:237‐251.
   Muratovska, A. and Eccles, M.R. 2004. Conjugate for efficient delivery of short interfering RNA (siRNA) into mamalian cells. FEBS Lett. 558:63‐68.
   Nitin, N., Santangelo, P.J., Kim, G., Nie, S., and Bao, G. 2004. Peptide‐linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucl. Acids Res. 32:e58.
   Pooga, M., Soomets, U., Hällbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J.‐X., Xu, X.‐J., Wiesenfeld‐Hallin, Z., Hökfelt, T., Bartfai, T., and Langel, Ü. 1998. Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16:857‐861.
   Potocky, T.B., Menon, A.K., and Gellman, S.H. 2003. Cytoplasmic and nuclear delivery of a TAT‐derived peptide and a β‐peptide after endocytic uptake into HeLa cells. J. Biol. Chem. 278:50188‐50194.
   Prater, C.E. and Miller, P. 2004. 3′‐Methylphosphonate‐modified oligo‐2′‐O‐methytlribonucleotides and their Tat peptide conjugates: Uptake and stability in mouse fibroblasts in culture. Bioconjug. Chem. 15:498‐507.
   Richard, J.‐P., Melikov, K., Vivès, E., Ramos, C., Verbeure, B., Gait, M.J., Chernomordik, L.V., and Lebleu, B. 2003. Cell‐penetrating peptides. A re‐evaluation of the mechanism of cellular uptake. J. Biol. Chem. 278:585‐590.
   Shi, F. and Hoekstra, D. 2004. Effective intracellular delivery of oligonucleotides in order to make sense of antisense. J. Control Release 97:189‐209.
   Stetsenko, D.A., Arzumanov, A.A., Korshun, V.A., and Gait, M.J. 2000. Peptide conjugates of oligonucleotides as enhanced antisense agents: A review. Mol. Biol. (Russ.) 34:852‐859.
   Thierry, A.R., Vivès, E., Richard, J.‐P., Prevot, P., Martinand‐Mari, C., Robbins, I., and Lebleu, B. 2003. Cellular uptake and intracellular fate of antisense oligonucleotides. Curr. Opin. Mol. Ther. 5:133‐138.
   Tripathi, S., Chaubey, B., Ganguly, S., Harris, D., Casale, R.A., and Pandey, P.K. 2005. Anti‐HIV‐1 activity of anti‐TAR polyamide nucleic acid conjugated with various membrane transducing peptides. Nucl. Acids Res. 33:4345‐4356.
   Turner, J.J., Arzumanov, A.A., and Gait, M.J. 2005a. Synthesis, cellular uptake and HIV‐1 Tat‐dependent trans‐activation inhibition activity of oligonucleotide analogues disulphide‐conjugated to cell‐penetrating peptides. Nucl. Acids Res. 33:27‐42.
   Turner, J.J., Ivanova, G.D., Verbeure, B., Williams, D., Arzumanov, A., Abes, S., Lebleu, B., and Gait, M.J. 2005b. Cell‐penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV‐1 Tat‐dependent trans‐activation in cells. Nucl. Acids Res. 33:6837‐6849.
   Vivès, E. and Lebleu, B. 1997. Selective coupling of a highly basic peptide to an oligonucleotide. Tetrahedron Lett. 38:1183‐1186.
   Wadia, J.S. and Dowdy, S.F. 2002. Protein transduction technology. Curr. Opin. Biotechnol. 13:52‐56.
   Zamecnik, P.C. and Stephenson, M.L. 1978. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. U.S.A. 75:280‐284.
   Zatsepin, T.S., Turner, J.J., Oretskaya, T.S., and Gait, M.J. 2005. Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents. Curr. Pharm. Des. 11:3639‐3654.
   Ziegler, A., Nervi, P., Dürrenberger, M., and Seelig, J. 2005. The cationic cell‐penetrating peptide CPPTat derived from the HIV‐1 protein TAT is rapidly transported into living fibroblasts: Optical, biphysical, and metabolic evidence. Biochemistry 44:138‐148.
   Zorko, M. and Langel, U. 2005. Cell‐penetrating peptides: Mechanism and kinetics of cargo delivery. Adv. Drug Deliv. Rev. 57:529‐545.
   Zubin, E.M., Romanova, E.A., and Oretskaya, T.S. 2002. Modern methods for the synthesis of peptide‐oligonucleotide conjugates. Russ. Chem. Rev. 71:239‐264.
PDF or HTML at Wiley Online Library