RNA Aptamers and Spiegelmers: Synthesis, Purification, and Post‐Synthetic PEG Conjugation

Stefanie Hoffmann1, Johannes Hoos1, Sven Klussmann1, Stefan Vonhoff1

1 NOXXON Pharma AG, Berlin, Germany
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.46
DOI:  10.1002/0471142700.nc0446s46
Online Posting Date:  September, 2011
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Abstract

This unit describes the solid‐phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1‐ to 4‐mmol scale with subsequent conjugation to PEG using the L‐RNA spiegelmer NOX‐E36 as an example. Following synthesis and two‐step deprotection, the crude oligonucleotide is purified by preparative reversed‐phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino‐modified oligonucleotide is reacted with NHS‐ester‐activated PEG, and the oligonucleotide‐PEG conjugate is obtained after preparative AX‐HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in‐process and quality controls. Curr. Protoc. Nucleic Acid Chem. 46:4.46.1‐4.46.30. © 2011 by John Wiley & Sons, Inc.

Keywords: solid phase RNA synthesis; preparative RP/AX‐HPLC purification; oligonucleotide‐PEG conjugation; Spiegelmer NOX‐E36

     
 
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Table of Contents

  • Introduction
  • Strategic Planning: Process Overview
  • Basic Protocol 1: Synthesis of Amino‐Modified RNA: NOX‐E36 pre‐PEG
  • Basic Protocol 2: Pegylation of Amino‐Modified NOX‐E36
  • Support Protocol 1: Analytical AX‐HPLC Method for Amino‐Modified and Pegylated Oligonucleotides
  • Support Protocol 2: Analytical RP‐HPLC Method for Amino‐Modified and Pegylated Oligonucleotides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Amino‐Modified RNA: NOX‐E36 pre‐PEG

  Materials
  • Amidites [ChemGenes, Innovassynth (http://www.innovassynth.com), Sigma‐Aldrich‐Proligo, ThermoFisher]:
    • L‐rABz phosphoramidite (5′‐O‐DMT‐2′‐TBDMS‐N6‐benzoyl‐L‐adenosine 3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite (mol. wt., 988.2 g/mol)
    • L‐rCAc phosphoramidite (5′‐O‐DMT‐2′‐TBDMS‐N4‐acetyl‐L‐cytidine 3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite (mol. wt., 902.1 g/mol)
    • L‐rGibu phosphoramidite (5′‐O‐DMT‐2′‐TBDMS‐N2‐isobutyryl‐L‐guanosine 3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite (mol. wt. 970.2 g/mol)
    • L‐rU phosphoramidite (5′‐O‐DMT‐2′‐TBDMS‐L‐uridine 3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite (mol. wt. 861.1 g/mol)
    • MMT‐protected C6 aminolinker phosphoramidite (mol. wt. 571.8 g/mol) [e.g., American International Chemical (http://www.aicma.com), ChemGenes, Sigma‐Aldrich‐Proligo, ThermoFisher]
  • Acetonitrile for DNA synthesis (specification, < 30 ppm water)
  • Drying bags or molecular sieves (American International Chemical or ChemGenes)
  • Detritylation solution: 10% dichloroacetic acid (DCA) in toluene; prepare by adding 1 L DCA to a graduated 10‐L Schott bottle, adding toluene to the 10‐L mark, and mixing
  • Activator solution: 0.6 M 5‐ethyl thiotetrazole (ETT; mol. wt. 130.2) in acetonitrile; prepare by adding 78.1 g ETT to a graduated 2‐L Schott bottle, adding acetonitrile to the 1‐L mark, and mixing until complete dissolution (dry over drying bags or molecular sieves for 24 hr before use)
  • Capping solutions:
    • CAP A: 20% N‐methyl imidazole (NMI), 30% lutidine, 50% acetonitrile
    • CAP B: 25% acetic anhydride in acetonitrile
  • Oxidizer: 0.05 M iodine in pyridine/water (9:1); higher‐concentration solutions of iodine can also be used without a change to the protocol
  • CPG: L‐rGibu loaded CPG with following specifications: pore size, 600 ű10%; loading, 70 to 80 µmol/g; density, 0.3 g/mL±10% (e.g., PrimeSynthesis, http://www.primesynthesis.com/); for aptamers exceeding 50 nucleotides a loading of 30 to 70 µmol/g and a pore size of 700 to 1000 Å is recommended, depending on the length of the oligonucleotide
  • 20% N,N‐diethylamine in acetonitrile: prepare by mixing 200 mL of N,N‐diethylamine and 800 mL of acetonitrile in a 2‐L Schott bottle
  • 40% (v/v) methylamine, aqueous
  • 50% (v/v) ethanol, aqueous
  • Dimethylsulfoxide (DMSO)
  • Triethylamine
  • Triethylamine trishydrofluoride (Sigma‐Aldrich)
  • Source 30RPC resin (GE Healthcare)
  • RP‐HPLC Buffer A (see recipe)
  • RP‐HPLC Buffer B (see recipe)
  • 20% (v/v) ethanol
  • 80% (v/v) acetic acid
  • Liquid N 2
  • 1‐, 2‐, 5‐, and 10‐L Schott bottles for preparation of amidite, activator, and detritylation solution
  • Shaker to fit 1‐L and 2‐L Schott bottles
  • ÄktaPilot100 synthesizer with adjustable column, 5.9‐cm i.d. (GE Healthcare)
  • 100‐mL and 1‐L measuring cylinder
  • 1‐L G1 fritted‐glass filtration funnel
  • 5‐L suction flask
  • Vacuum pump inert to methylamine (with exhaust line connected to gas washer absorbing methylamine)
  • 2‐L round‐bottom flasks
  • Butyl rubber septa to fit the 2‐L round‐bottom flasks
  • Rotary evaporator
  • Shaker (to fit 1‐L and 2‐L Schott bottles)with incubation hood which can be heated to 60°C
  • FineLine100 column GE Healthcare, or similar chromatography column with 100‐mm inner diameter
  • ÄktaPurifier 100 (GE Healthcare) or similar chromatography platform with UV detector, delivering up to 100 mL/min per pump
  • Fraction collector with 50‐mL Falcon tubes
  • 500‐mL dropping funnel
  • Laboratory‐scale ultrafiltration system:
    • For solutions < 1 L containing < 50 kOD of oligonucleotide—Labscale TFF System equipped with three Pellicon XL 50‐cm2 membranes (Millipore), or equivalent.
    • For solutions > 1 L containing > 50 kOD of oligonucleotide—Sartojet UF System equipped with two Sartocon Slice cassettes, 0.1‐m2 (Sartorius), or equivalent
  • Conductivity meter
  • Spectrophotometer
  • Bench‐top lyophilizer with up to 4‐kg ice capacity, which can hold round‐bottom flasks and lyophilization flask in which Falcon tubes can be placed (e.g., Christ, http://www.martinchrist.de/; GEA, http://www.gea‐ps.com/)
  • Dewar flasks capable of holding up to 2‐L round‐bottom flasks
  • Lyophilization flasks (available from lyophilizer suppliers)
  • Additional reagents and equipment for analytical AX‐HPLC ( protocol 3), RP‐HPLC ( protocol 4), and quantitation of nucleic acids by UV absorption (unit 5.2)

Basic Protocol 2: Pegylation of Amino‐Modified NOX‐E36

  Material
  • Solution of 100 kOD of amino‐modified NOX‐E36 in 35 mL of Milli‐Q water: this solution can be prepared by dissolving the lyophilized NOX‐E36 ( protocol 1) in 80% of the target volume of water in a 500‐mL round‐bottom flask, measuring the concentration by UV analysis (unit 5.2), and adjusting to the final concentration by addition of water; alternatively, the concentrated desalted solution of amino‐modified NOX‐E36 in water as prepared in protocol 1 can be diluted with Milli‐Q water to a target concentration of ∼2900 OD/mL in a 500‐mL round‐bottom flask
  • Theorell‐Stenhagen buffer, pH 8.2 to 8.5 and stock solution (see recipe)
  • N,N‐dimethylformamide (DMF)
  • 40‐kDa PEG‐NHS ester: NOX‐E36 conjugated to a 40‐kDa PEG (JenKem, http://jenkemusa.net); other 40‐kDa PEG‐NHS esters can be used equivalently
  • 1 M hydrochloric acid
  • AX‐HPLC Buffer A (see recipe)
  • AX‐HPLC Buffer B (see recipe)
  • Source 30Q anion‐exchange resin (GE Healthcare)
  • 20% (v/v) ethanol
  • 500‐mL and 2‐L round bottom flasks
  • Hot plate with magnetic stirrer and egg‐shaped stir‐bar (4‐ to 5‐cm length), or overhead stirrer
  • ÄktaPurifier100 (GE Healthcare) or similar chromatography platform with UV detector delivering up to 100 mL/min per pump
  • Heat exchanger capable of heating solvents at a flow of 100 mL/min to 60°C (e.g., Timberline TL‐600 mobile phase pre‐heater; http://www.timberlineinstruments.com/)
  • FineLine100 column (GE Healthcare) or similar chromatography column with 100‐mm inner diameter
  • Fraction collector holding 250‐mL Schott bottles (alternatively, 250‐mL fractions can be collected manually)
  • Additional reagents and equipment for desalting ( protocol 1, steps 24 to 27) and lyophilization ( protocol 1, steps 28 to 30) of amino‐modified NOX‐E36

Support Protocol 1: Analytical AX‐HPLC Method for Amino‐Modified and Pegylated Oligonucleotides

  Materials
  • Water, HPLC grade or Milli‐Q water, or equivalent
  • Tris (ultrapure)
  • Ethylendiaminetetraacetic acid disodium salt dihydrate (EDTA; for molecular biology)
  • Sodium perchlorate, monohydrate (analytical grade)
  • Acetonitrile (HPLC‐S gradient grade)
  • Hydrochloric acid (analytical grade)

Support Protocol 2: Analytical RP‐HPLC Method for Amino‐Modified and Pegylated Oligonucleotides

  Materials
  • Water, HPLC grade or Milli‐Q water, or equivalent
  • Acetonitrile (HPLC‐S gradient grade)
  • TEAA Buffer Solution 2 M (HPLC grade, e.g., Biosolve)
  • Triethylamine (minimum 99%)
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Figures

Videos

Literature Cited

Literature Cited
   Bonilla, J.V. and Srivatsa, G.S. 2011. Handbook of Analysis of Oligonucleotides and Related Products. CRC Press, Boca Raton, Florida.
   Bouchard, P.R., Hutabarat, R.M., and Thompson, K.M. 2010. Discovery and development of therapeutic aptamers. Annu. Rev. Pharmacol. Toxicol. 50:237–257.
   Eulberg, D., Purschke, W., Anders, H.‐J., Selve, N. and Klussmann, S. 2008. Spiegelmer NOX‐E36 for renal diseases. In Therapeutic Oligonucleotides ( J. Kurreck, ed.) pp. 200‐221. Biomolecular Sciences Series of the Royal Society of Chemistry (RSC), London.
   Hermanson, G.T. 2008. Bioconjugate Techniques. Academic Press, Burlington, Mass.
   Klussmann, S. 2006. The Aptamer Handbook. Wiley‐VCH, Weinheim, Germany.
   Mascini, M. 2009. Aptamers in Bioanalysis. John Wiley & Sons, Hoboken, N.J.
   Phillips, A.T. and Signs, M.W. 2005. Desalting, concentration, and buffer exchange by dialysis and ultrafiltration. Curr. Protoc. Protein Sci. 38:4.4.1‐4.4.15.
   Rey, L. and May, J.C.. 2007. Freeze‐Drying/Lyophilisation of Pharmaceutical and Biological Products. Informa Healthcare, New York.
   Sanghvi, Y.S. and Schulte, M. 2004. Therapeutic oligonucleotides: The state‐of‐the‐art in purification technologies. Curr. Opin. Drug Discov. Dev 7:765–776.
   Sproat, B.S. 2005. RNA synthesis using 2′‐O‐(tert‐butyldimethylsilyl) protection. Methods Mol. Biol. 288:17‐32.
   Sproat, B., Colonna, F., Mullah, B., Tsou, D., Andrus, A., Hampel, A., and Vinayak, R. 1995. An efficient method for the isolation and purification of oligoribonucleotides, Nucleosides and Nucleotides 14:255‐273.
   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis and purification of RNA and ribozymes, Nucleic Acid Res. 23:2677‐2684.
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