Native Chemical Ligation of Hydrolysis‐Resistant 3′‐NH‐Cysteine‐Modified RNA

Anna‐Skrollan Geiermann1, Ronald Micura2

1 Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo‐ku, Tokyo, 2 Institute of Organic Chemistry, Center for Chemistry and Biomedicine, Leopold Franzens University, Innsbruck, Austria
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.64
DOI:  10.1002/0471142700.nc0464s62
Online Posting Date:  September, 2015
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Abstract

Hydrolysis‐resistant RNA‐peptide conjugates that contain a 3′‐NH linkage between the adenosine ribose and the C‐terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid‐phase oligonucleotide synthesis and bioconjugation is described here. The key step is native chemical ligation (NCL) of 3′‐NH‐cysteine‐modified RNA to highly soluble peptide thioesters. These hydrolysis‐resistant 3′‐NH‐peptide‐modified RNAs, containing the universally conserved 3′‐CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle. © 2015 by John Wiley & Sons, Inc.

Keywords: oligonucleotide‐peptide conjugates; native chemical ligation; nucleoside modification; RNA solid‐phase synthesis; peptides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the Cysteine‐Modified Solid Support rA3′NH‐Cys
  • Alternate Protocol 1: Preparation of the Cysteine‐Modified Solid Support dA3′NH‐Cys
  • Support Protocol 1: Preparation of General Building Blocks
  • Basic Protocol 2: Synthesis, Purification, and Characterization of 3′‐NH‐Cysteine‐Modified RNA‐Oligoribonucleotides
  • Alternate Protocol 2: Synthesis of 3′‐NH‐Cysteine‐Modified DNA‐Oligonucleotides
  • Basic Protocol 3: Native Chemical Ligation of 3′‐NH‐Cysteine‐Modified Oligonucleotides
  • Basic Protocol 4: Desulfurization of Cysteine‐Containing 3′‐NH‐Peptidyloligonucleotides
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of the Cysteine‐Modified Solid Support rA3′NH‐Cys

  Materials
  • 9‐(β‐D‐Arabinofuranosyl)‐9 H‐adenine (Metkinen Chemistry, cat. no. 203‐05)
  • Triphenylphosphine, purity ≥98.5%
  • Argon gas
  • Dioxane, anhydrous, 99.8% pure
  • N,N‐Dimethylformamide (DMF), anhydrous, 99.8% pure
  • Diisopropyl azodicarboxylate (DIAD), 98% pure
  • Methanol (MeOH)
  • Silica gel (SiO 2), pore size 60 Å, 70‐230 mesh
  • Methylene chloride (dichloromethane; CH 2Cl 2), purity >99.5%
  • Lithium azide (LiN 3; see recipe)
  • N,N‐Di‐n‐butylformamide dimethyl acetal (see Support Protocol)
  • Pyridine, anhydrous
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), purity ≥97.0%
  • Tetrabutylammonium nitrate, 97% pure
  • Toluene
  • Half‐saturated aqueous sodium bicarbonate (NaHCO 3) solution
  • Half‐saturated aqueous sodium chloride (NaCl) solution
  • Sodium sulfate (Na 2SO 4)
  • 4‐Dimethylaminopyridine (DMAP), purum, purity ≥98.0%
  • N‐Ethyldiisopropylamine (iPr 2NEt), purity ≥98.0%
  • Trifluoromethanesulfonyl chloride, purity ≥99.0%
  • Acetone
  • 18‐Crown‐6, purity ≥98.0%
  • Potassium trifluoroacetate, 98% pure
  • N‐Allyloxycarbonyl‐S‐(tert‐butylthio)‐L‐cysteine pentafluorophenylester [alloc‐Cys(StBu)‐OPfp; see Support Protocol]
  • Adipic acid dipentafluorophenylester (see Support Protocol)
  • Custom Primer Support 200 Amino (GE Healthcare, cat. no. 28‐9229‐46)
  • 0.5 M 4‐Dimethylaminopyridine (DMAP) in acetonitrile (capping solution A)
  • 2:3:5 v/v/v Acetic anhydride/2,4,6‐trimethylpyridine/acetonitrile (capping solution B)
  • Acetonitrile, HPLC grade
  • 100‐mL, 250‐mL 3‐neck round‐bottom flasks
  • Stir bar
  • Rubber septa
  • Reflux condenser
  • Hot plate magnetic stirrer
  • Oil bath
  • Water bath
  • Disposable syringes and needles
  • 5‐mL, 10‐mL, 50‐mL, 100‐mL, 250‐mL round‐bottom flasks
  • Rotary evaporator
  • Splash bulb
  • Glass chromatography column
  • TLC plates (POLYGRAM SIL G/UV 254 plates with 0.2 mm silica gel layer and fluorescent indicator, Machery‐Nagel)
  • Short‐wave UV lamp
  • Vacuum pump
  • Filter paper
  • Büchner funnel
  • 250‐mL, 500‐mL separatory funnels
  • 100‐mL, 250‐mL Erlenmeyer flasks
  • 1000‐μL, 500‐μL Hamilton syringes
  • Büchner funnel with frit (funnel capacity: 2 mL, 4‐8 μm porosity)
  • Additional reagents and equipment for performing thin‐layer chromatography ( appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 1: Preparation of the Cysteine‐Modified Solid Support dA3′NH‐Cys

  Materials
  • 9‐(3′‐Amino‐2′,3′‐dideoxy‐β‐D‐ribofuranosyl)adenine (Metkinen Chemistry, cat. no. 203‐11)
  • N‐Allyloxycarbonyl‐S‐(tert‐butylthio)‐L‐cysteine pentafluorophenylester [alloc‐Cys(StBu)‐OPfp; see Support Protocol]
  • Argon gas
  • N,NDimethylformamide (DMF), anhydrous
  • Pyridine, anhydrous
  • Tertbutyl(chloro)dimethylsilane (TBDMS‐Cl), 97%
  • Methanol (MeOH)
  • Methylene chloride (dichloromethane, CH 2Cl 2), purity >99.5%
  • 5% (w/v) Citric acid in H 2O
  • Saturated aqueous sodium bicarbonate (NaHCO 3) solution
  • Saturated aqueous sodium chloride (NaCl) solution
  • Sodium sulfate (Na 2SO 4)
  • Silica gel
  • 3‐(2,6‐Dioxotetrahydro‐2 H‐pyran‐3‐yl)propanoic acid (see Support Protocol)
  • Pentafluorophenol (HOPfp), purity ≥99.0%
  • 1‐Hydroxybenzotriazole monohydrate (HOBt), 98% pure
  • N,N′‐Diisopropylcarbodiimide (DIC), 99% pure
  • Acetone
  • Custom Primer Support 200 Amino (GE Healthcare, cat. no. 28‐9229‐46)
  • 0.5 M 4Dimethylaminopyridine (DMAP) in acetonitrile (Capping solution A)
  • Acetic anhydride/2,4,6‐trimethylpyridine/acetonitrile (2:3:5 v/v/v) (Capping solution B)
  • Acetonitrile, HPLC grade
  • 10‐mL, 25‐mL, 50‐mL, 100‐mL round‐bottom flasks
  • Stir bar
  • Rubber septa
  • Hot plate magnetic stirrer
  • Rotary evaporator
  • 250‐mL separatory funnel
  • 100‐mL Erlenmeyer flask
  • Glass chromatography column
  • TLC plates (POLYGRAM SIL G/UV 254 plates with 0.2 mm silica gel layer and fluorescent indicator, Machery‐Nagel)
  • Short‐wave UV lamp
  • Vacuum pump
  • Reflux condenser
  • Oil bath
  • Filter paper
  • Büchner funnel
  • Büchner funnel with frit (funnel capacity: 2 mL, 4‐8 μm porosity)
  • Additional reagents and equipment for performing thin‐layer chromatography ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Preparation of General Building Blocks

  Materials
  • Dibutylamine, purity ≥98%
  • N,N‐Dimethylformamide dimethyl acetal, 94%
  • S‐(Tert‐butylthio)‐L‐cysteine [H‐Cys(StBu)‐OH]
  • Sodium carbonate (Na 2CO 3)
  • Tetrahydrofuran (THF)
  • Diallyl pyrocarbonate, purity ≥95.0%
  • Diethyl ether
  • Hydrochloric acid (HCl), concentrated
  • Methylene chloride (dichloromethane, CH 2Cl 2)
  • Sodium sulfate (Na 2SO 4)
  • Pentafluorophenol (HOPfp), purity ≥99.0%
  • 4‐Dimethylaminopyridine (DMAP), purity ≥98.0%
  • N,N′‐Diisopropylcarbodiimide (DIC), 99%
  • 10% (w/w) Hydrochloric acid (HCl)
  • Half‐saturated aqueous sodium bicarbonate (NaHCO 3) solution
  • Saturated aqueous sodium chloride (NaCl) solution
  • Adipic acid
  • N‐(3‐Dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC·HCl), purity ≥99.0%
  • N,N‐Dimethylformamide (DMF), anhydrous, 99.8%
  • Argon gas
  • Silica gel (SiO 2), pore size 60 Å, 70‐230 mesh
  • Pentane‐1,3,5‐tricarboxylic acid
  • Paraffin oil
  • NaOH solution, 20% (w/v)
  • 1,2Dichloroethane, anhydrous
  • Thionyl chloride (SOCl 2)
  • Hexane
  • 25‐mL, 100‐mL round‐bottom flasks
  • Stir bar
  • Reflux condenser
  • Oil bath
  • Distillation head with a condenser sealed to a Vigreux column (e.g., micro distillation head with Vigreux indents, Sigma‐Aldrich)
  • Thermometer and adapter
  • Pig type receiving adapter
  • Ice bath
  • 1000‐μL Hamilton syringe
  • Magnetic stirrer
  • 100‐mL separatory funnel
  • 100‐mL Erlenmeyer flask
  • pH indicator
  • Rotary evaporator
  • Glass chromatography column
  • TLC plates (POLYGRAM SIL G/UV 254 plates with 0.2 mm silica gel layer and fluorescent indicator; Machery‐Nagel)
  • 100‐mL 3‐neck round‐bottom flask
  • Bubble counter filled with paraffin oil
  • Wash bottles
  • Büchner funnel
  • Additional reagents and equipment for performing thin‐layer chromatography ( appendix 3D) and column chromatography (appendix 3e)

Basic Protocol 2: Synthesis, Purification, and Characterization of 3′‐NH‐Cysteine‐Modified RNA‐Oligoribonucleotides

  Materials
  • Phosphoramidites:
    • 5′‐DMT‐2′‐TOM‐riboguanosine (N2‐acetyl) phosphoramidite (ChemGenes, cat. no. ANP‐3203)
    • 5′‐DMT‐2′‐TOM‐riboadenosine (N6‐acetyl) phosphoramidite (ChemGenes, cat. no. ANP‐3201)
    • 5′‐DMT‐2′‐TOM‐ribocytidine (N4‐acetyl) phosphoramidite (ChemGenes, cat. no. ANP‐3202)
    • 5′‐DMT‐2′‐TOM‐ribouridine phosphoramidite (ChemGenes, cat. no. ANP‐3205)
  • 5‐(Benzylthio)‐1 H‐tetrazol (BTT; Biosolve, cat. no. 027024)
  • Acetonitrile (ACN; HPLC‐grade quality), anhydrous (dry over activated molecular sieves overnight)
  • 4% (v/v) Dichloroacetic acid in 1,2‐dichloroethane (Detritylation solution)
  • 10 mM Iodine in acetonitrile/2,4,6‐trimethylpyridine/H 2O (10:1:5 v/v/v) (Oxidation solution; see recipe)
  • 0.5 M 4‐Dimethylaminopyridine (DMAP) in acetonitrile (Capping solution A)
  • 2:3:5 (v/v/v) Acetic anhydride/2,4,6‐trimethylpyridine/acetonitrile (Capping solution B)
  • 1,2‐Dichloroethane
  • rA3′NH‐Cys solid support (see protocol 1)
  • Argon
  • Methylene chloride (dichloromethane; CH 2Cl 2), anhydrous
  • Dimethylamine borane [(H 3C) 2NH·BH 3], 97%
  • Tetrakis(triphenylphosphine)palladium [(Ph 3P) 4Pd], 99%
  • N,N‐Dimethylformamide (DMF), anhydrous, 99.8%
  • 0.5% (w/v) Sodium diethyldithiocarbamate hydrate in DMF
  • Methanol (MeOH)
  • 20% (v/v) Piperidine in acetonitrile
  • 8 M Methylamine in Ethanol (EtOH)
  • 40% (v/v) Methylamine in water
  • 50% (v/v) Ethanol in water
  • 1 M Tetrabutylammonium fluoride trihydrate (TBAF) in THF/H 2O (9:1 v/v)
  • 1 M Triethylammonium acetate (TEAA) buffer, pH 6.4
  • Ethanol (EtOH)
  • Nanopure water: for the preparation of HPLC eluants and for all oligonucleotide solutions; 18 MΩ/cm
  • Eluant A: 25 mM Tris·Cl buffer, pH 8.0, 6 M urea:filter through a cellulose acetate filter (0.2‐μm pore size, Sartorius) prior to use)
  • Eluant B: 25 mM Tris·Cl buffer, pH 8.0, 0.5 M NaClO 4, 6 M urea:filter through a cellulose acetate filter (0.2‐μm pore size, Sartorius) prior to use
  • 0.1 M Triethylammonium bicarbonate (TEAB) buffer
  • Ethylenediaminetetraacetic acid (EDTA)
  • Eluant C: 8.6 mM triethylamine (TEA), 100 mM 1,1,1,3,3,3‐hexafluoroisopropanol (HFIP) in H 2O, pH 8.3: filter through a cellulose acetate filter (0.2 μm pore size, Sartorius) prior to use
  • Eluant D: methanol (MeOH)
  • Molecular sieves (4 Å), activated
  • Automated DNA synthesizer (e.g., Pharmacia Gene Assembler Plus; Applied Biosystems)
  • Syringes
  • Synthesis column for 1.5‐μmol scale
  • Vacuum pump
  • 10‐mL and 100‐mL round‐bottom flasks
  • Rubber septa
  • Büchner funnel with frit (funnel capacity: 2 mL, 4‐8 μm porosity)
  • 2‐mL Screw‐capped tube or vial
  • Centrifuge
  • Rotary evaporator
  • Purification system: e.g., ÄKTAprime plus, GE Healthcare; HiPrep 26/10 desalting column (2.6 × 10 cm, Sephadex G25, GE Healthcare)
  • HPLC system: e.g., HPLC Äkta Basic, GE Healthcare; DNAPac PA‐100 (4 × 250 mm, Dionex); DNAPac PA‐100 (9 × 250 mm, Dionex)
  • C18 SepPakPlus cartridge (Waters/Millipore)
  • Lyophilizer
  • LC‐ESI mass spectrometer (e.g., Finnigan LCQ Advantage MAX ion trap, 2.1 × 100 mm Amersham μRPC C2/C18 column)
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C)

Alternate Protocol 2: Synthesis of 3′‐NH‐Cysteine‐Modified DNA‐Oligonucleotides

  Additional Materials (also see protocol 4)
  • dA3′NH‐Cys solid support
  • Tetrabutylammonium fluoride trihydrate (TBAF)
  • Acidic acid
  • Tetrahydrofuran (THF)
  • N,N‐Dimethylformamide (DMF), anhydrous, 99.8%
  • Methanol (MeOH)
  • Methylene chloride (dichloromethane; CH 2Cl 2), anhydrous
  • Phosphoramidites:
    • 5′‐DMT‐2′‐deoxyguanosine (N2‐isobutyryl) phosphoramidite (ChemGenes, cat. no.ANP‐5553)
    • 5′‐DMT‐2′‐deoxyadenosine (N6‐benzoyl) phosphoramidite (ChemGenes, cat. no. ANP‐5551)
    • 5′‐DMT‐2′‐deoxycytidine (N4‐acetyl) phosphoramidite (ChemGenes, cat. no. ANP‐5560)
    • 5′‐DMT‐2′‐thymidine phosphoramidite (ChemGenes cat. no. ANP‐5554)
  • 32% (v/v) Aqueous ammonia
  • 8 M Methylamine in Ethanol (EtOH)
  • Ethanol (EtOH)
  • Nanopure water
  • 10‐mL round bottom flasks
  • Büchner funnel with frit (funnel capacity: 2 mL, 4‐8 μm porosity)Vacuum apparatus
  • 2‐mL screw‐capped tube or vial
  • Rotary evaporator

Basic Protocol 3: Native Chemical Ligation of 3′‐NH‐Cysteine‐Modified Oligonucleotides

  Materials
  • 3′‐Cysteinyloligonucleotide (see protocol 4 or protocol 5)
  • Ligation buffer: 7 M urea, 1 M Tris·Cl buffer, pH 8.0, 0.1 M TCEP, 2% (v/v) thiophenol
  • Argon gas
  • 0.6 M Sodium acetate buffer, pH 5.0
  • Eluant A: 25 mM Tris·Cl buffer, pH 8.0, 6 M urea; filter through a cellulose acetate filter (0.2 μm pore size, Sartorius) prior to use
  • Eluant B: 25 mM Tris·Cl buffer, pH 8.0, 0.5 M NaClO 4, 6 M urea; filter through a cellulose acetate filter (0.2 μm pore size, Sartorius) prior to use
  • 2‐mL Eppendorf reaction tube
  • Sonicator
  • HPLC system:e.g., HPLC Äkta Basic (GE Healthcare), DNAPac PA‐100 (4 × 250 mm, Dionex)
NOTE: Use nanopure water for the preparation of buffer solutions and HPLC eluants (18 MΩ/cm).

Basic Protocol 4: Desulfurization of Cysteine‐Containing 3′‐NH‐Peptidyloligonucleotides

  Additional Materials (also see protocol 6)
  • 0.1 M aqueous Ammonium citrate solution
  • 1 M Sodium phosphate buffer, pH 7.5
  • 0.5 M Tris(carboxyethyl)phosphine (TCEP) solution, pH 7.3
  • 20 mM Glutathione solution
  • 100 mM 2,2′‐Azobis(2‐methylpropionamidine)dihydrochloride solution (V50; Sigma‐Aldrich)
  • Argon
  • Eluant A: 25 mM Tris·Cl buffer, pH 8.0, 6 M urea
  • Eluant B: 25 mM Tris·Cl buffer, pH 8.0, 0.5 M NaClO 4, 6 M urea
  • 2‐mL Eppendorf reaction tube
  • Eppendorf MiniSpin
  • Centrifugal concentrator (Vivaspin 500, membrane: 3000 MWCO PES, product number: VS0191, Sartorius)
  • Lyophilizer
  • Sonicator
  • HPLC system: e.g., HPLC Äkta Basic (GE Healthcare), DNAPac PA‐100 (4 × 250 mm, Dionex)
NOTE: Use nanopure water for the preparation of buffer solutions and HPLC eluants (18 MΩ/cm).
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Literature Cited

Literature Cited
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Key References
  Moroder et al., 2009. See above.
  The authors describe the syntheses of 3′’‐amino‐3′‐deoxyadenosine‐functionalized support rA3′‐NH and 3′‐amino‐2′,3′‐dideoxyadenosine‐functionalized support dA3′‐NH for the automated solid‐phase synthesis of nonhydrolyzable RNA‐peptide conjugates. The presented approach employs automated standard peptide synthesis on the solid support followed by automated oligonucleotide assembly.
  Dawson, P., Muir, T., Clark‐Lewis, I., and Kent, S. 1994. Synthesis of proteins by native chemical ligation. Science 266:776‐779.
  The authors report the first practical method to ligate large unprotected peptide fragments and demonstrate the utility of native chemical ligation.
  Haase, C., Rohde, H., and Seitz, O. 2008. Native Chemical Ligation at Valine. Angew. Chem. Int. Ed. 47:6807‐6810.
  In this work the authors present native chemical ligation at valine using the precursor β,β‐dimethylcysteine (penicillamine). Metal‐free desulfurization is achieved using a water‐soluble radical starter which abstracts a hydrogen atom from the cysteine thiol group, which is then reduced with tris(carboxyethyl)phosphine (TCEP) to form an alkyl radical. Glutathione serves here as hydrogen source to donate a hydrogen atom to the alkyl radical to furnish valine.
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