Using Triple‐Helix‐Forming Peptide Nucleic Acids for Sequence‐Selective Recognition of Double‐Stranded RNA

Dziyana Hnedzko1, Samwel K. Cheruiyot1, Eriks Rozners1

1 Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.60
DOI:  10.1002/0471142700.nc0460s58
Online Posting Date:  September, 2014
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Abstract

Non‐coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double‐helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence‐selective recognition of double‐stranded RNA (dsRNA) using triple‐helix‐forming peptide nucleic acids (PNAs) that bind in the major grove of the RNA helix. The strategy developed uses chemically modified nucleobases, such as 2‐aminopyridine (M), which enables strong triple‐helical binding under physiologically relevant conditions, and 2‐pyrimidinone (P) and 3‐oxo‐2,3‐dihydropyridazine (E), which enable recognition of isolated pyrimidines in the purine‐rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid‐phase synthesis, HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included. Curr. Protoc. Nucleic Acid Chem. 58:4.60.1‐4.60.23. © 2014 by John Wiley & Sons, Inc.

Keywords: double‐stranded RNA; triple helix; peptide nucleic acids; PNA; isothermal titration calorimetry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of M, 2‐Aminopyridine PNA Monomer
  • Basic Protocol 2: Synthesis of P, 2‐Pyrimidinone PNA Monomer
  • Basic Protocol 3: Synthesis of E, 3‐Oxo‐2,3‐Dihydropyridazine PNA Monomer
  • Basic Protocol 4: Solid‐Phase PNA Synthesis
  • Basic Protocol 5: Purification and Quantification of PNA and RNA
  • Basic Protocol 6: RNA Binding Affinity by Isothermal Titration Calorimetry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of M, 2‐Aminopyridine PNA Monomer

  Materials
  • 2‐(6‐Chloro‐3‐pyridinyl)acetic acid ethyl ester [synthesized from commercially available 2‐(6‐chloro‐3‐pyridinyl) acetonitrile (Matrix Scientific, CAS 39891‐09‐3) using a catalytic amount of concentrated sulfuric acid in ethanol as described by Burns et al. ( )]
  • Tert‐butyl carbamate (Matrix Scientific, cat. no. 074774)
  • 9,9‐Dimethyl‐4,5‐bis(diphenylphosphino)xanthene (Matrix Scientific, cat. no. 073845)
  • Tris(dibenzylideneacetone)dipalladium (Acros Organics, cat. no. 318770050)
  • Cesium carbonate
  • Anhydrous tetrahydrofuran
  • Nitrogen gas (N 2)
  • 10% (w/v) aqueous ammonium acetate
  • Ethyl acetate
  • Brine: sodium chloride (NaCl), saturated aqueous solution
  • Silica gel (SiliaFlash P60, 230 to 400 mesh; Silicycle, cat. no. R12030B)
  • Methanol
  • Dichloromethane (CH 2Cl 2)
  • Sodium hydroxide (NaOH)
  • Citric acid
  • Hexanes
  • PNA backbone (7; Wojciechowski and Hudson, )
  • 3, 4‐dihydro‐3‐hydroxy‐4‐oxo‐1,2,3‐benzotriazin‐4(3H)‐one (HOBt)
  • Anhydrous dimethylformamide
  • N,N‐dicyclohexylcarbodiimide (DCC)
  • Sodium hydrogen carbonate (NaHCO 3), 5% aqueous solution
  • N‐ethylaniline (TCI America, cat. no. E0059)
  • Potassium hydrogen sulfate (KHSO 4), saturated aqueous solution
  • 25‐mL, 50‐mL, and 250‐mL round‐bottom flasks
  • Magnetic stirrer and stir bar
  • Reflux condenser
  • 125‐ and 250‐mL separatory funnels
  • 60‐mL fritted glass funnel
  • Rotary evaporator equipped with Vacuubrand PC520 NT Dry Chemistry Vacuum System (http://www.vacuubrand.com)
  • 2.5 × 15‐ and 2.5 × 8‐cm chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of P, 2‐Pyrimidinone PNA Monomer

  Materials
  • PNA backbone (8; Wojciechowski and Hudson, )
  • (2‐oxo‐1(2H)‐pyrimidinyl)acetic acid (ChemBridge, cat. no. 4028397)
  • 3,4‐dihydro‐3‐hydroxy‐4‐oxo‐1,2,3‐benzotriazin‐4(3H)‐one (HOBt)
  • Anydrous dimethylformamide (DMF)
  • N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide (EDC; Oakwood Products, cat. no. 024810)
  • Hydrochloric acid (HCl), 1 M aqueous solution
  • Sodium bicarbonate (NaHCO 3), 5% aqueous solution
  • Sodium chloride (NaCl), saturated aqueous solution (brine)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (SiliaFlash P60, 230 to 400 mesh; Silicycle, cat. no. R12030B)
  • Methanol
  • Dichloromethane (CH 2Cl 2)
  • Nitrogen gas (N 2)
  • 10% palladium on carbon (10% Pd/C; Strem Chemicals, cat. no. 46‐1900)
  • Hydrogen gas (H 2)
  • Celite 545
  • Ethyl acetate
  • 250‐ and 500‐mL round‐bottom flasks
  • Magnetic stirrer and stir bar
  • 1000‐mL separatory funnel
  • Rotary evaporator equipped with Vacuubrand PC520 NT Dry Chemistry Vacuum System (http://www.vacuubrand.com)
  • 2.5 × 15–cm chromatography column

Basic Protocol 3: Synthesis of E, 3‐Oxo‐2,3‐Dihydropyridazine PNA Monomer

  Materials
  • PNA backbone (8; Wojciechowski and Hudson, )
  • 3‐(6‐(benzyloxy)pyridazin‐3‐ylamino)propanoic acid (12; Eldrup et al., )
  • N,N‐dicyclohexylcarbodiimide (DCC)
  • Sodium hydrogen carbonate (NaHCO 3), 5% aqueous solution
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (SiliaFlash P60, 230 to 400 mesh; Silicycle, cat. no. R12030B)
  • Methanol
  • Dichloromethane (CH 2Cl 2)
  • Nitrogen gas (N 2)
  • 10% palladium on carbon (10% Pd/C; Strem Chemicals, cat. no. 46‐1900)
  • Hydrogen gas (H 2)
  • Celite 545
  • 250‐mL round‐bottom flask
  • Rotary evaporator
  • 250‐mL separatory funnel
  • 60‐mL fritted glass funnel
  • 2.5 × 15‐ and 2.5 × 8‐cm chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Basic Protocol 4: Solid‐Phase PNA Synthesis

  Materials
  • NovaSyn TG Sieber resin (commercially available from NovaBiochem, functionalized at 0.2 mmol/g)
  • Fmoc‐PNA‐T‐OH (commercially available from Link Technologies)
  • Fmoc‐PNA‐M(Boc)‐OH ( protocol 1)
  • Fmoc‐PNA‐E‐OH ( protocol 2)
  • Fmoc‐PNA‐P‐OH ( protocol 3)
  • N‐methyl‐2‐pyrrolidone (NMP)
  • Reagents for oligonucleotide synthesis:
    • Deblocking solution (see recipe)
    • Activator solution (see recipe)
    • Base solution (see recipe)
    • Capping solution (see recipe)
    • Cleavage Cocktail (see recipe)
  • Anhydrous dimethylformamide (DMF)
  • 2‐(1H‐7‐azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyl uronium hexafluorophosphate (HATU)
  • Fmoc‐Lys(Boc)‐OH (commercially available from NovaBiochem)
  • Diisopropylethylamine (DIPEA)
  • Anhydrous diethyl ether
  • HPLC‐grade H 2O
  • Synthesis columns for the Expedite 8909 (commercially available from Glen Research)
  • Expedite 8909 monomer vials (Glen Research or Link Technologies)
  • Expedite 8909 synthesizer (ABI)
  • UV spectrophotometer
  • Two 1‐mL syringes
  • Small vials with silicone rubber septa
  • 2‐mL microcentrifuge tubes

Basic Protocol 5: Purification and Quantification of PNA and RNA

  Materials
  • PNA sample ( protocol 4)
  • HPLC‐grade H 2O
  • Mobile phase A (see recipe)
  • Mobile phase B (see recipe)
  • Crude sample of RNA target (e.g., Thermo Fisher Scientific)
  • Deprotection buffer (provided by RNA vendor)
  • Mobile phase C (see recipe)
  • Mobile phase D (see recipe)
  • 0.2‐µm syringe filters
  • HPLC system consisting of gradient‐capable pumps, column oven, and UV detector
  • Waters XBridge Prep C‐18 column (5 µm, 10 mm × 150 mm)
  • Rotary evaporator equipped with Vacuubrand PC520 NT Dry Chemistry Vacuum System (http://www.vacuubrand.com)
  • Freeze‐dry system with SpeedVac capability
  • UV spectrophotometer
  • 60°C water bath
  • Additional reagents and equipment for mass spectrometry of oligonucleotides (unit 10.1)

Basic Protocol 6: RNA Binding Affinity by Isothermal Titration Calorimetry

  Materials
  • Stock solution of purified and quantified target RNA ( protocol 5)
  • Stock solution of purified and quantified PNA sample ( protocol 5)
  • Phosphate buffer, pH 7.4 [see recipe; add a small stir bar to the vial with ITC buffer and degas by stirring under moderate vacuum (∼80 torr) for 10 to 15 min)]
  • HPLC‐grade H 2O
  • 2‐mL microcentrifuge tubes
  • SpeedVac evaporator
  • Isothermal titration microcalorimeter suitable for small biological samples (cell volume 1 mL or smaller and minimum detectable heat 0.1 µJ or smaller; e.g., TA Instruments NanoITC 2G instrument
  • ITC titration syringe and burette (e.g., TA instruments)
  • NanoAnalyze software (TA Instruments)
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Figures

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Literature Cited

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