A High‐Throughput Process for the Solid‐Phase Purification of Synthetic DNA Sequences

Andrzej Grajkowski1, Jacek Cieślak1, Serge L. Beaucage1

1 Laboratory of Biological Chemistry, Food and Drug Administration, Silver Spring, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.17
DOI:  10.1002/cpnc.31
Online Posting Date:  June, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′‐terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′‐hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid‐phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid‐phase capture of the nucleobase‐ and phosphate‐deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full‐length DNA sequences are first washed away from the capture support; the solid‐phase purified DNA sequences are then released from this support upon reaction with tetra‐n‐butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid‐phase‐purified DNA sequences exceeds 98%. The simulated high‐throughput and scalability features of the solid‐phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc.

Keywords: cost‐effective process; high‐throughput capability; large scale purification; synthetic DNA sequences; solid‐phase purification

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

Table of Contents

  • Introduction
  • Basic Protocol 1: General Procedure for the Preparation of a Capture Support
  • Basic Protocol 2: Preparation of Chemical Linkers for the Solid‐Phase Capture of DNA Sequences and for Determining the Surface Density of Aminooxy Functions Covalently Bound to Support 3
  • Basic Protocol 3: Preparation of 5′‐Functionalized Deoxyribonucleosides and Deoxyribonucleoside Phosphoramidites for Solid‐Phase Capture of DNA Sequences
  • Support Protocol 1: Solid‐Phase Synthesis of 5′‐Functionalized Phosphorothioate and Native DNA Sequences
  • Basic Protocol 4: General Procedure for the Solid‐Phase Capture and Release of Phosphorothioate and Native DNA Sequences
  • Alternate Protocol 1: Scalable Solid‐Phase Purification of a Relevant DNA Sequence
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: General Procedure for the Preparation of a Capture Support

  Materials
  • 7‐Oxooctanoic acid (1; Aldrich)
  • 1,1′‐Carbonyldiimidazole (CDI; Aldrich)
  • Tetrahydrofuran, dry (THF; Acros)
  • 3‐Aminopropyl silica gel (∼1 mmol NH 2, Aldrich)
  • Acetonitrile (MeCN; Aldrich)
  • Triethylamine (Et 3N; Aldrich)
  • Cap Mix A (Glen research)
  • Cap Mix B (Glen Research)
  • O,O′‐1,3‐Propanediylbishydroxylamine dihydrochloride (Aldrich)
  • Magnetic stir bars
  • 25‐ and 50‐mL round‐bottom flasks
  • 8‐mesh Drierite with indicator (Aldrich)
  • Drierite guard tube
  • Reflux condenser (LabGlass)
  • 5‐ and 10‐mL Luer‐tipped glass syringes
  • Magnetic stirrer
  • 15‐mL, 30‐mL fritted glass vacuum filtration Buchner funnel of coarse porosity (ChemGlass)
  • 100‐mL Erlenmeyer flasks
  • 50‐mL glass cylinder
  • Vacuum line (ChemGlass)
  • Rubber septa for 14/20‐glass joints
  • Temperature controlled orbital shaker (Thomas Scientific)
  • 21‐G stainless steel syringe needles
  • High‐vacuum oil pump (Edwards)
  • 10‐mL screw‐cap glass vials with caps

Basic Protocol 2: Preparation of Chemical Linkers for the Solid‐Phase Capture of DNA Sequences and for Determining the Surface Density of Aminooxy Functions Covalently Bound to Support 3

  Materials
  • N,N′‐Dimethylethylenediamine (Aldrich)
  • γ‐Butyrolactone (Aldrich)
  • Acetonitrile (MeCN; Aldrich)
  • Silica gel (60‐Å, 230 to 400 mesh; EMD)
  • Methanol (MeOH, Fisher Scientific)
  • Chloroform (CHCl 3;3 Fisher Scientific)
  • 5,5‐Dimethyldihydrofuran‐2‐one (ACS Scientific)
  • 7‐Oxooctanoic acid (1; Aldrich)
  • Tetrahydrofuran, dry (THF; Acros)
  • Pyridine, dry (Acros)
  • 4‐Methoxytrityl chloride (Aldrich)
  • Anhydrous sodium sulfate (Aldrich)
  • Toluene (Fisher Scientific)
  • Triethylamine (Et 3N; Aldrich)
  • Dimethyl sulfoxide (DMSO; Aldrich)
  • Dichloromethane (CH 2Cl 2; Fisher Scientific)
  • 3% Trichloroacetic acid in dichloromethane (Deblock solution, Glen Research)
  • 1,1′‐Carbonyldiimidazole (CDI; Aldrich)
  • Deuterated dimethylsulfoxide (DMSO‐d 6; Aldrich)
  • Phosphomolybdic acid (Aldrich)
  • 4‐ and 25‐mL screw‐cap glass vials with caps
  • Dry heat block (VWR)
  • 25‐ 50‐ and 100‐mL round‐bottom flasks (Kontes)
  • High‐vacuum oil pump (Edwards)
  • Rubber septa for 14/20 glass joints (Aldrich)
  • Rotary evaporator connected to a vacuum pump (Büchi)
  • Pasteur pipettes (VWR)
  • 5 × 20‐cm glass chromatography columns (LabGlass)
  • 13 × 100‐mm disposable glass tubes
  • 2.5 × 7.5‐cm TLC plates precoated with a 250‐μm layer of silica gel 60 F254 (EMD)
  • Magnetic stir bars
  • Magnetic stirrer
  • Reflux condenser (LabGlass)
  • 8‐mesh Drierite with indicator (Aldrich)
  • Drierite guard tube
  • Pipettor (Corning)
  • Magnetic hot‐plate stirrer (Corning)
  • 5‐ and 10‐mL Luer‐tipped glass syringes
  • 21‐G stainless steel syringe needles
  • 50‐mL glass cylinder
  • 100‐mL Separatory funnel15‐mL, 30‐mL fritted glass vacuum filtration Buchner funnel of coarse porosity (ChemGlass)
  • Vacuum line (LabGlass)
  • 50‐ and 100‐mL Erlenmeyer flasks
  • Temperature controlled orbital shaker (Thomas Scientific)
  • 10‐mL volumetric flask (Thomas Scientific)
  • Hand‐held UV 254 lamp (UVP)
  • UV/vis spectrophotometer (Agilent Technologies)
  • NMR spectrometer (Bruker)
  • 1‐mL pipette tips (Fisher Scientific)
  • Additional reagents and equipment for column chromatography (Meyers, , appendix 3E) and TLC (Meyers and Meyers, , appendix 3D)

Basic Protocol 3: Preparation of 5′‐Functionalized Deoxyribonucleosides and Deoxyribonucleoside Phosphoramidites for Solid‐Phase Capture of DNA Sequences

  Materials
  • N‐(2‐(4‐hydroxy‐N,4‐dimethylpentanamido)ethyl)‐N‐methyl‐7‐oxooctanamide (6b; see protocol 2)
  • 1H‐Imidazole (Aldrich)
  • Argon
  • N,N‐Dimethylformamide, dry (DMF; Acros)
  • N,N‐Diisopropylethylamine (Aldrich)
  • Dichlorodiisopropylsilane (Aldrich)
  • Isopropyl ether (Aldrich)
  • N6‐Benzoyl‐2′‐deoxyadenosine (7a; ChemGenes)
  • Sodium bicarbonate (NaHCO 3; Fisher Scientific)
  • Ethyl acetate (EtOAc; VWR)
  • Chloroform (CHCl 3; Fisher Scientific)
  • Silica gel (60‐Å, 230 to 400 mesh; EMD)
  • Methanol (MeOH; Fisher Scientific)
  • N4‐Benzoyl‐2′‐deoxycytidine (7b; ChemGenes)
  • N2‐Isobutyryl‐2′‐deoxyguanosine (7c; ChemGenes)
  • 2′‐Deoxythymidine (7d; ChemGenes)
  • Dichloromethane, dry (CH 2Cl 2; Acros)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • Anhydrous sodium sulfate (Fisher Scientific)
  • Benzene (C 6H 6; Aldrich)
  • Triethylamine (Et 3N; Aldrich)
  • Deuterated benzene (C 6D 6; Aldrich)
  • Acetone (Fisher Scientific)
  • Liquid nitrogen
  • Deuterated dimethyl sulfoxide (DMSO‐d 6, Aldrich)
  • Magnetic stir bars
  • 50‐ and 100‐mL round‐bottom flasks (Kontes)
  • 1‐ and 10‐mL glass syringes
  • Pipettor (Corning)
  • 1‐mL pipet tips (Fisher Scientific)
  • Rubber septa for 14/20 glass joints (Aldrich)
  • Wet ice bath
  • Magnetic stirrer
  • Dry ice‐isopropyl ether bath
  • 100‐mL separatory funnel
  • 50‐mL glass cylinder
  • Rotary evaporator connected to a vacuum pump (Büchi)
  • Pasteur pipettes (VWR)
  • Chromatography glass columns (LabGlass)
  • 13×100‐mm disposable glass tubes
  • Hand‐held UV 254 lamp (UVP)
  • UV/vis spectrophotometer (Agilent Technologies)
  • NMR spectrometer (Bruker)
  • 2.5 × 7.5‐cm TLC plates precoated with a 250‐μm layer of silica gel 60 F254 (EMD)
  • 21‐G stainless steel syringe needles
  • 100‐mL Erlenmeyer flask (Kontes)
  • 30‐mL fritted glass vacuum filtration Buchner funnel of coarse porosity (ChemGlass)
  • In‐house vacuum line
  • Dry ice‐acetone bath
  • Lyophilizer
  • Dewark flasks
  • Additional reagents and equipment for thin‐layer chromatography (TLC; Meyers and Meyers, , appendix 3D)

Support Protocol 1: Solid‐Phase Synthesis of 5′‐Functionalized Phosphorothioate and Native DNA Sequences

  Additional Materials (also see protocol 3)
  • Long‐chain alkylamine controlled‐pore glass (500 Å LCAA‐CPG or 2000 Å LCAA‐CPG) support functionalized with 1‐μmol 5′‐O‐(4,4′‐ dimethoxytrityl)‐2′‐deoxythymidine as the leader nucleoside (Glen Research).
  • 5′‐O‐(4,4′‐dimethoxytrityl)‐dABz, ‐dGiBu, ‐dCBz and ‐dT phosphoramidite monomers (ChemGenes)
  • 5′‐Functionalized deoxyribonucleoside phosphoramidites 9a‐d (see protocol 3)
  • Anhydrous acetonitrile diluent (MeCN, Glen Research)
  • Reagents for oligonucleotide synthesis (all available from Glen Research):
    • 0.45 M 1 H‐tetrazole or 0.25 M 5‐ethythio‐1 H‐tetrazole in MeCN
    • Cap A solution: acetic anhydride in tetrahydrofuran (THF)/pyridine
    • Cap B solution: 1‐methylimidazole in THF
    • Oxidation solution: 0.05 M 3 H‐1,2‐benzodithiol‐3‐one 1,1‐dioxide in MeCN or 0.02 M iodine in THF/pyridine/water
    • Deblocking solution: 3% trichloroacetic acid in CH 2Cl 2
  • Concentrated aqueous ammonia (NH 3; Aldrich)
  • DNA/RNA synthesizer (394 DNA/RNA synthesizer, Applied Biosystems)
  • 4‐mL screw cap glass vial with caps (Fisher Scientific)
  • 1‐mL glass syringes
  • 1‐mL plastic syringes

Basic Protocol 4: General Procedure for the Solid‐Phase Capture and Release of Phosphorothioate and Native DNA Sequences

  Materials
  • Capture solid support 3 (see protocol 1)
  • Acetonitrile (MeCN or CH 3CN; Aldrich)
  • Triethylamine (Et 3N; Aldrich)
  • Tetra‐n‐butyl ammonium chloride (Aldrich)
  • 5′‐functionalized DNA sequences 10a‐f (see protocol 4Support Protocol)
  • Concentrate aqueous ammonia (NH 3; Aldrich)
  • Dimethyl sulfoxide (DMSO; Aldrich)
  • Tetra‐n‐butylammonium fluoride hydrate (Aldrich)
  • Methoxytrimethylsilane
  • Peroxide‐free tetrahydrofuran (THF; Aldrich)
  • 15‐mL fritted glass vacuum filtration Buchner funnel of coarse porosity (ChemGlass)
  • 100‐mL Erlenmeyer flask (Fisher Scientific)
  • 1‐ and 10‐mL plastic syringes (Fisher Scientific)
  • In‐house vacuum line
  • 4‐mL screw‐cap glass vial with caps (Fisher Scientific)
  • Spatula
  • Orbital shaker
  • 5 μm Supelcosil 25 cm × 4.6 mm LC‐18 S column (Supelco)
  • RP‐HPLC (Agilent Technologies)
  • Rotary evaporator connected to a vacuum pump (Büchi)
  • 1‐ and 10‐mL glass syringes (Fisher Scientific)
  • Heath block (VWR)
  • 25‐mL round‐bottom flask
  • 1.5‐mL microcentrifuge tubes
  • Pasteur pipettes (VWR)
  • Centrifuge
  • Pipettor (Corning)
  • SpeedVac system
  • 1‐mL pipet tips (Fisher Scientific)
  • High vacuum oil pump

Alternate Protocol 1: Scalable Solid‐Phase Purification of a Relevant DNA Sequence

  Materials
  • 5′‐Functionalized phosphorothioate DNA sequences (10a, protocol 4Support Protocol)
  • Tetra‐n‐butylammonium chloride (Aldrich)
  • Dimethyl sulfoxide (DMSO; Aldrich)
  • Capture support 3 ( protocol 1)
  • Triethylamine (Et 3N; Aldrich)
  • Acetonitrile (MeCN or CH 3CN, Aldrich)
  • Dimethyl sulfoxide, dry (DMSO; Acros)
  • Tetra‐n‐butylammonium fluoride hydrate (Aldrich)
  • Methoxytrimethylsilane (Aldrich)
  • Peroxide‐free tetrahydrofuran (THF; Aldrich)
  • DNA/RNA synthesizer (394 DNA/RNA synthesizer, Applied Biosystems)
  • 25‐ and 100‐mL round‐bottom flasks
  • 5‐mL plastic syringes
  • Rotary evaporator connected to a vacuum pump (Büchi)
  • 5‐ and 10‐mL glass syringes
  • 20‐mL screw‐cap glass vial with caps (Kimble)
  • Temperature‐controlled orbital shaker (Thomas Scientific)
  • 15‐mL fritted glass vacuum filtration Buchner funnel of coarse porosity (ChemGlass)
  • 100‐mL Erlenmeyer flask
  • In‐house vacuum line
  • Heat block (VWR)
  • Pipettor (Corning)
  • 10‐mL glass cylinder
  • Vortex mixer (VWR)
  • 1‐mL pipette tips (Fisher Scientific)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Andrus, A. & Kuimelis, R. G. (2001). Polyacrylamide gel electrophoresis (PAGE) of synthetic nucleic acids. Current Protocols in Nucleic Acid Chemistry. 1, 10.4.1–10.4.10. doi: 10.1002/0471142700.nc1004s01.
  Beaucage, S. L. (1993). Oligodeoxyribonucleotides synthesis–Phosphoramidite approach. In S, Agrawal (Ed). Methods in Molecular Biology. (Vol. 20: Protocols for Oligonucleotides and Analogs pp. 33–61). Totowa: Humana Press, Inc.
  Beaucage, S. L., & Caruthers, M. H. (1981). Deoxynucleoside phosphoramidites–A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters, 22, 1859–1862. doi: 10.1016/S0040‐4039(01)90461‐7.
  Beller, C., & Bannwarth, W. (2005). Noncovalent attachment of nucleotides by fluorous‐fluorous interactions: Application to a simple purification principle for synthetic DNA fragments. Helvetica Chimica Acta, 88, 171–179. doi: 10.1002/hlca.200490291.
  Cieślak, J., Ausín, C., Grajkowski, A., & Beaucage, S. L. (2013). The 2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl group for the 2′‐hydroxyl protection of ribonucleosides in the solid‐phase synthesis of RNA sequences. Chemistry European Journal, 19, 4623–4632. doi: 10.1002/chem.201204235.
  Cieślak, J., Grajkowski, A., Ausín, C., Gapeev, A., & Beaucage, S. L. (2012). Permanent or reversible conjugation of 2′‐O‐ or 5′‐O‐aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences. Nucleic Acids Research, 40, 2312–2329. doi: 10.1093/nar/gkr896.
  Crooke, S. T. (2004). Antisense strategies. Current Molecular Medicine, 4, 465–487. doi: 10.2174/1566524043360375.
  Dandapani, S. (2006). Recent applications of fluorous separation methods in organic and bioorganic chemistry. QSAR & Combinatorial Science, 25, 681–688. doi: 10.1002/qsar.200640051.
  Defrancq, E., & Lhomme, J. (2001). Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides. Bioorganic & Medicinal Chemistry Letters, 11, 931–933. doi: 10.1016/S0960‐894X(01)00108‐1.
  Dorsett, Y., & Tuschl, T. (2004). siRNAs: Applications in functional genomics and potential as therapeutics. Nature reviews Drug Discovery, 3, 318–329. doi: 10.1038/nrd1345.
  Eleuteri, A., Capaldi, D. C., Krotz, A. H., Cole, D. L., & Ravikumar, V. T. (2000). Pyridinium trifluoroacetate/N‐methylimidazole as an efficient activator for oligonucleotide synthesis via the phosphoramidite method. Organic Process Research & Development, 4, 182–189. doi: 10.1021/op9900378.
  Ellington, A., & Pollard, J. D. Jr. (2001). Introduction to the synthesis and purification of oligonucleotides. Current Protocols in Nucleic Acid Chemistry. 00, A.3C.1–A.3C.22. doi: 10.1002/0471142700.nca03cs00.
  Fang, S., & Bergstrom, D. E. (2003a). Reversible biotinylation phosphoramidite for 5′‐end‐labeling, phosphorylation, and affinity purification of synthetic oligonucleotides. Bioconjugate Chemistry, 14, 80–85. doi: 10.1021/bc025626o.
  Fang, S., & Bergstrom, D. E. (2003b). Fluoride cleavable biotinylation phosphoramidite for 5′‐end‐labeling and affinity purification of synthetic oligonucleotides. Nucleic Acids Research, 31, 708–715. doi: 10.1093/nar/gkg130.
  Fang, S., & Bergstrom, D. E. (2003c). Reversible biotinylation of the 5′‐terminus of oligodeoxyribonucleotides and its application in affinity purification. Current Protocols in Nucleic Acid Chemistry, 14, 4.20.1–4.20.17. doi: 10.1002/0471142700.nc0420s14.
  Fang, S., & Bergstrom, D. E. (2004). Reversible 5′‐end biotinylation and affinity purification of synthetic RNA. Tetrahedron Letters, 45, 7987–7990. doi: 10.1016/j.tetlet.2004.09.019.
  Fang, S., & Fueangfung, S. (2010). Scalable synthetic oligodeoxynucleotide purification with use of a catching by polymerization, washing, and releasing approach. Organic Letters, 12, 3720–3723. doi: 10.1021/ol101316g.
  Fang, S., Fueangfung, S., Lin, X., Zhang, X., Mai, W., Bi, L., & Green, S. A. (2011). Synthetic oligodeoxynucleotide purification by polymerization of failure sequences. Chemical Communications, 47, 1345–1347. doi: 10.1039/C0CC04374E.
  Fina, N. J., & Edwards, J. O. (1973). The alpha effect. A review. International Journal of Chemistry. Kinetics, 5, 1–26. doi: 10.1002/kin.550050102.
  Forget, D., Boturyn, D., Defrancq, E., Lhomme, J., & Dumy, P. (2001a). Highly efficient synthesis of peptide‐oligonucleotide conjugates: Chemoselective oxime and thiazolidine formation. Chemistry European journal, 7, 3976–3984. doi: 10.1002/1521‐3765(20010917)7:18%3c3976::AID‐CHEM3976%3e3.0.CO;2‐X.
  Forget, D., Renaudet, O., Boturyn, D., Defrancq, E., & Dumy, P. (2001c). 3′‐Oligonucleotides conjugation via chemoselective oxime bond formation. Tetrahedron Letters, 42, 9171–9174. doi: 10.1016/S0040‐4039(01)02017‐2.
  Forget, D., Renaudet, O., Defrancq, E., & Dumy, P. (2001b). Efficient preparation of carbohydrate‐oligonucleotide conjugates (COCs) using oxime bond formation. Tetrahedron Letters, 42, 7829–7832. doi: 10.1016/S0040‐4039(01)01682‐3.
  Grajkowski, A., Cieślak, J., & Beaucage, S. L. (2016). Solid‐phase purification of synthetic DNA sequences. The Journal of Organic Chemistry, 8, 6165–6175. doi: 10.1021/acs.joc.6b01020.
  Iyer, R. P., Phillips, L. R., Egan, W., Regan, J. B., & Beaucage, S. L. (1990). The automated synthesis of sulfur‐containing oligodeoxyribonucleotides using 3H‐1,2‐benzodithiol‐3‐one 1,1‐dioxide as a sulfur‐transfer reagent. The Journal of Organic Chemistry, 55, 4693–4698. doi: 10.1021/jo00302a039.
  Katajisto, J., Virta, P., & Lönnberg, H. (2004). Solid‐phase synthesis of multiantennary oligonucleotide glycoconjugates utilizing on‐support oximation. Bioconjugate Chemistry, 15, 890–896. doi: 10.1021/bc049955n.
  Kawasaki, A. M., Casper, M. D., Prakash, T. P., Manalili, S., Sasmor, H., Manoharan, M., & Cook, P. D. (1999). Synthesis, hybridization, and nuclease resistance properties of 2′‐O‐aminooxyethyl (2′‐O‐AOE) modified oligonucleotides. Tetrahedron Letters, 40, 661–664. doi: 10.1016/S0040‐4039(98)02498‐8.
  Meyers, C. (2001). Column chromatography. Current Protocols in Nucleic Acid Chemistry, 3, A.3E.1–A.3E.7. doi: 10.1002/0471142700.nca03es03.
  Meyers, C., & Meyers, D. (2008). Thin‐layer chromatography. Current Protocols in Nucleic Acid Chemistry, 34, A.3D.1–A.3D.13. doi: 10.1002/0471142700.nca03ds34.
  Mishra, R., Mishra, S., & Misra, K. (2006). Synthesis and application of fluorous‐tagged oligonucleotides. Chemistry Letters, 35(10), 1184–1185. doi: 10.1246/cl.2006.1184.
  Morvan, F., Sanghvi, Y. S., Perbost, M., Vasseur, J.‐J., & Bellon, L. (1996). Oligonucleotide mimics for antisense therapeutics: Solution phase and automated solid‐support synthesis of MMI linked oligomers. Journal of the American Chemical Society, 118, 255–256. doi: 10.1021/ja9533959.
  Pearson, W. H., Berry, D. A., Stoy, P., Jung, K‐Y., & Sercel, A. D. (2005). Fluorous affinity purification of oligonucleotides. The Journal of Organic Chemistry, 70, 7114–7122. doi: 10.1021/jo050795y.
  Pokharel, D., Yuan, Y., Fueangfung, S., & Fang, S. (2014). Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization. RSC Advances, 4, 8746–8757. doi: 10.1039/C3RA46986G.
  Salo, H., Virta, P., Hakala, H., Prakash, T. P., Kawasaki, A. M., Manoharan, M., & Lönnberg, H. (1999). Aminooxy functionalized oligonucleotides: Preparation, on‐support derivatization, and postsynthetic attachment to polymer support. Bioconjugate Chemistry, 10, 815–823. doi: 10.1021/bc990021m.
  Sinha, N. D., Biernat, J., McManus, J., & Köster, H. (1984). Polymer support oligonucleotide synthesis XVIII: Use of β‐cyanoethyl‐N,N‐dialkylamino‐/N‐morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Research, 12, 4539–4557. doi: 10.1093/nar/12.11.4539.
  Sinha, N.D. and Jung, K.E. (2015). Analysis and purification of synthetic nucleic acids using HPLC. Current Protocols in Nucleic Acid Chemistry, 61, 10.5.1–10.5.39. doi: 10.1002/0471142700.nc1005s61
  Sproat, B. S., Rupp, T., Menhardt, N., Keane, D., & Beijer, B. (1999). Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag. Nucleic Acids Research, 27, 1950–1955 doi: 10.1093/nar/27.8.1950.
  Swiderski, P. M., Bertrand, E. L., & Kaplan, B. E. (1994). Polystyrene reverse‐phase ion‐pair chromatography of chimeric ribosymes. Analytical Biochemistry, 216, 83–88. doi: 10.1006/abio.1994.1011.
  Trader, D. J., & Carlson, E. E. (2011). Siloxyl ether functionalized resins for chemoselective enrichment of carboxylic acids. Organic Letters, 13, 5652–5655. doi: 10.1021/ol202376m.
  Trevisiol, E., Renard, A., Defrancq, E., & Lhomme, J. (1997). The oxyamino‐aldehyde coupling reaction: An efficient method for the derivatization of oligonucleotides. Tetrahedron Letters, 38, 8687–8690. doi: 10.1016/S0040‐4039(97)10335‐5.
  Wei, X. (2013). Coupling activators for the oligonucleotide synthesis via phosphoramidite approach. Tetrahedron, 69, 3615–3637. doi: 10.1016/j.tet.2013.03.001.
  Wilk, A., Grajkowski, A., Chmielewski, M. K., Phillips, L. R., & Beaucage, S. L. (2001). Deoxyribonucleoside phosphoramidites. Current Protocols in Nucleic Acid Chemistry, 4, 2.7.1–2.7.12. doi: 10.1002/0471142700.nc0207s04.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library