Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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(Ot Bu)‐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 NH₄ HCO₃ 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 t Bu, 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. NH₄ OAc (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

Figure 4.28.1 Formation of disulfide‐linked conjugates of peptides and antisense cargoes.

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Figure 4.28.2 HPLC chromatogram of conjugate formation from peptide RQIKIWFQNRRMKWKKGGC with (pys)S‐(CH₂ )₆ ‐5′‐2′‐ O ‐Me/LNA [C UC CC A GGC UC A]‐3′‐fluorescein; peaks (i) salts and formamide, (ii) excess peptide, (iii) conjugate product, (iv) unconjugated oligo(pys). The solid trace is at 280 nm and the dashed trace is at 480 nm, which identifies the fluorescein label on the oligonucleotide.

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Figure 4.28.3 MALDI‐TOF mass spectra of GRKKKRRQRRRPC(S‐)‐S‐(CH₂ )₆ ‐5′‐2′‐ O ‐Me/LNA[CUC CCA GGC UCA]‐3′‐fluorescein conjugate: (A ) purified and (B ) pure conjugate with 0.5 eq. peptide added.

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Figure 4.28.4 RP‐HPLC of (A ) the synthesis of RRRRRRRQIKIWFQNRRMKWKKGGC‐CK[CTCCCAGGCTCAGATC]_PNA KKK and (B ) analysis of the purified product.

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Figure 4.28.5 MALDI‐TOF mass spectrum of the conjugate described in Figure .

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Videos

Literature Cited

Literature Cited
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Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited