Synthesis of Oligodeoxynucleotides with 5′‐Caps Binding RNA Targets

Simone Egetenmeyer1, Clemens Richert1

1 Institute for Organic Chemistry, University of Stuttgart, Stuttgart, Germany
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.53
DOI:  10.1002/0471142700.nc0453s51
Online Posting Date:  December, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5′‐O‐terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of L‐prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step‐wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap‐bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis. Curr. Protoc. Nucleic Acid Chem. 51:4.53.1‐4.53.21. © 2012 by John Wiley & Sons, Inc.

Keywords: oligonucleotides; phosphoramidite; peptides; controlled pore glass; hybridization probes

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of a Molecular Cap Building Block
  • Alternate Protocol 1: Synthesis of 2′‐Oligodeoxyribonucleotides with 5′‐Cap Build Up on Solid Support
  • Support Protocol 1: Synthesis of N‐Fmoc‐L‐Prolinol Linker Phosphoramidite
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Synthesis of a Molecular Cap Building Block

  Materials
  • 1‐H‐Benzotriazole, 98% (Fluka)
  • Argon or nitrogen (dry)
  • Thionyl chloride, 99% (Sigma)
  • Oxolinic acid (Sigma)
  • Glycine, 99% (Acros)
  • Acetonitrile (Fisher Scientific) HPLC‐grade, water content 0.009%
  • Triethylamine (TEA), HPLC‐grade
  • Hydrochloric acid, concentrated (37%)
  • Pyridine, anhydrous, dried over molecular sieve (4 Å)
  • N,N‐dimethylformamide (DMF), dried over molecular sieves (4 Å)
  • N,N‐diisopropylethylamine (DIEA), HPLC‐grade (Sigma)
  • 2‐(1‐H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyl uronium hexafluorophosphate (HBTU; Iris Biotech, http://www.iris‐biotech.de/)
  • L‐prolinol, 95% (Acros)
  • Methanol (MeOH), HPLC grade
  • Dichloromethane (CH 2Cl 2), HPLC grade
  • Sodium bicarbonate solution (NaHCO 3 in water), saturated
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (0.04‐ to 0.07‐mm mesh; Merck)
  • Diisopropyl ammonium tetrazolide (DIPAT), ultra pure (ChemGenes)
  • O‐2‐cyanothyl‐N,N,N′,N′‐tetraisopropylphosphordiamidite, 99% (ChemGenes)
  • n‐pentane, HPLC grade
  • Round‐bottom flasks (50 mL)
  • Magnetic stirring bars and plate
  • Rubber septa
  • Argon balloon attached to syringe and needle for pressure equilibration in a septum‐closed flask
  • Büchner funnel
  • Filter paper
  • Membrane vacuum pump
  • High‐vacuum oil pump (giving 0.1 mbar pressure)
  • Rotary evaporator
  • Silica gel TLC plates with 254‐nm UV indicator (Merck)
  • UV‐lamp, wavelengths 254 nm and 366 nm
  • 40°C water bath
  • 0°C ice/water bath
  • Flash chromatography column (diameter 2 cm)
  • Syringes (10 mL, 1 mL, 200 µL volume), dried under reduced pressure of membrane pump at 10 mbar

Basic Protocol 2:

  Materials
  • Controlled pore‐glass (CPG) 1000 Å pore size (Proligo) loaded with 5′‐O‐(4,4′‐dimethoxytrityl)‐N‐protected‐deoxyribonucleoside with a loading of 30 to 40 µmol/g—the nucleobase in the 2′‐deoxynucleosides may be N6‐benzoyladenine (dA(Bz)‐CPG), thymine (T‐CPG), N4‐benzoylcytosine (dC(Bz)‐CPG), or N2‐isobutyrylguanine (dG(iBu)‐CPG)
  • S.1 ( protocol 1)
  • Argon (Ar) and nitrogen (N 2) from gas cylinders
  • DCI activator solution (Proligo): 0.25 M 4,5‐dicyanoimidazole in acetonitrile
  • Oxidizer solution (Proligo): tetrahydrofuran/water/pyridine/iodine; 90.54/9.05/0.41/0.43 (v/v/v/w) for DNA synthesis
  • Acetonitrile (Fisher scientific) HPLC grade, water content < 0.009%
  • 27% (v/v) aqueous ammonia (Sigma)
  • Triethylammonium acetate (TEAA) buffer: 0.1 M TEAA, pH 7
  • Reaction vial, 1.5 mL, also known as “Eppendorf cups,” made of polypropylene (Neolab, http://www.neolab.info/)
  • Syringe and small‐diameter needle
  • Heat block or water bath (60°C)
  • High‐vacuum oil pump (giving 0.1 mbar pressure)
  • Lyophilizer
  • PTFE or PVDF syringe filter (0.45 µm pore size)
  • 250 × 4.6 mm column (Nucleosil 120‐5 C18, Macherey‐Nagel, http://www.mn‐net.com/)
  • Additional reagents and equipment for flash chromatography ( appendix 3D), MALDI‐TOF mass spectrometry (unit 10.10), DNA quantitation by spectrophotometry (unit 5.2), and reversed‐phase chromatography (unit 10.5)

Alternate Protocol 1: Synthesis of 2′‐Oligodeoxyribonucleotides with 5′‐Cap Build Up on Solid Support

  Materials
  • Controlled pore‐glass 1000 Å pore size (Proligo), loading 30 to 40 µmol/g, loaded with preassembled oligonucleotide after standard DNA synthesis (DMT‐off mode)
  • O‐2‐Cyanoethyl‐O‐[N‐fluorenylmethylcarboxyl)‐(S)‐pyrrolidin‐3‐methoxy]‐(diisopropylamino)phosphoramidite ( S.8; for synthesis, see protocol 4)
  • Argon (Ar)
  • DCI activator solution (Proligo): 0.25 M 4,5‐dicyanoimidazole in acetonitrile
  • Oxidizer solution (Proligo): tetrahydrofuran/water/pyridine/iodine 90.54/9.05/0.41/0.43 (v/v/v/w) for DNA synthesis
  • Acetonitrile (Fisher scientific) HPLC grade, water content < 0.009%
  • 27% aqueous ammonia (Sigma)
  • Piperidine
  • N,N‐Dimethylformamide (DMF)
  • Fmoc‐Gly‐OH (Merck Millipore, cat. no. 852001)
  • 2‐(1‐H‐Benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyl uronium hexafluorophosphate (HBTU, Iris Biotech, http://www.iris‐biotech.de/)
  • N,N‐Diisopropylethylamine (DIEA)
  • Oxolinic acid (Sigma)
  • Nitrogen source
  • High‐vacuum oil pump (giving 0.1 mbar pressure)
  • Wide‐neck 100‐mL round‐bottom flask
  • Reaction vial, 1.5 mL, also known as “Eppendorf cups,” made of polypropylene (Neolab, http://www.neolab.info/)
  • Syringe with small‐diameter needle
  • Heat block or water bath (55° and 60°C)
  • Lyophilizer
  • Additional reagents and equipment for MALDI‐TOF mass spectrometry (unit 10.10), spectrophotometric quantitation of DNA (unit 5.2), purification of oligonucleotide ( protocol 2, steps 13 to 15), and reversed‐phase chromatography (unit 10.5)

Support Protocol 1: Synthesis of N‐Fmoc‐L‐Prolinol Linker Phosphoramidite

  Materials
  • (S)‐N‐Fmoc‐prolinol, 98% (Acros)
  • Argon or nitrogen (dry)
  • Dichloromethane (CH 2Cl 2), dried over molecular sieves
  • N,N‐diisopropylethylamine (DIEA), HPLC grade (Sigma)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Brine (saturated aqueous NaCl)
  • Triethylamine (TEA)
  • Silica gel (0.04‐ to 0.07‐mm mesh; Merck)
  • n‐pentane, HPLC grade
  • Round‐bottom flasks (50 mL)
  • Rubber septa
  • Argon balloon attached to syringe and needle for pressure equilibration of septum‐sealed flask
  • Rotary evaporator
  • Vacuum pump
  • Flash chromatography column (diameter 2 cm)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D), and flash chromatography ( appendix 3E)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Ahlborn, C., Siegmund, K., and Richert, C. 2007. Isostable DNA. J. Am. Chem. Soc. 129:15218‐15232.
   Altman, R.K., Schwope, I., Sarracino, D.A., Tetzlaff, C.N., Bleczinski, C.F., and Richert, C. 1999. Selection of modified oligonucleotides with increased target affinity via MALDI‐monitored nuclease survival assays. J. Combin. Chem. 1:493‐508.
   Arar, K., Aubertin, A.‐M., Roche, A.‐C., Monsigny, M., and Mayer, R. 1995. Synthesis and antiviral activity of peptide‐oligonucleotide conjugates prepared by using Nα‐(bromoacetyl)peptides. Bioconjug. Chem. 6:573‐577.
   Besik, I. and Marky, L.A. 1999. DNA, RNA, and DNA/RNA oligomer duplexes: A comparative study of their stability, heat, hydration, and Mg2+ binding properties. J. Phys. Chem. 103:8759‐8767.
   Bleczinski, C.F. and Richert, C. 1999. Steroid‐DNA interactions increasing stability, sequence‐selectivity, DNA/RNA discrimination, and hypochromicity of oligonucleotide duplexes. J. Am. Chem. Soc. 121:10889‐10894.
   Delatorre, B.G., Avino, A., Tarrason, G., Piulats, J., Albericio, F., and Eritja, R. 1994. Stepwise solid‐phase synthesis of oligonucleotide‐peptide. Tetrahedron Lett. 35:2733‐2736.
   Dreef‐Tromp, C.M., van Dam, E.M.A., van ven Elst, H., van der Marel, G.A., and Van Boom, J.H. 1990. Solid‐phase synthesis of H‐Phe‐Tyr‐(pATAT)‐NH2: A nucleopeptide fragment from the nucleoprotein of bacteriophage ϕX174. Nucleic Acids Res. 18:6491‐6495.
   Dogan, Z., Paulini, R., Rojas Stütz, J.A., Narayanan, S., and Richert, C. 2004. 5′‐Tethered stilbene derivatives as fidelity‐ and affinity‐enhancing modulators of DNA duplex stability. J. Am. Chem. Soc. 126:4762‐4763.
   Egetenmeyer, S. and Richert, C. 2011. A 5′‐cap for DNA probes binding RNA target strands. Chem. Eur. J. 17:11813‐11827.
   Grünefeld, P. and Richert, C. 2004. Synthesis of a 1′‐aminomethylthymidine and oligodeoxyribonucleotides with 1′‐acylamidomethylthymidine residues. J. Org. Chem. 69:7543‐7551.
   Haralambidis, J., Duncan, L., and Tregear, G.W. 1987. The solid‐phase synthesis of oligonucleotides containing a 3′‐peptide moiety. Tetrahedron Lett. 28:5199‐5202.
   Harrison, J.G. and Balasubramanian, S. 1998. Synthesis and hybridization analysis of a small library of peptide‐oligonucleotide conjugates. Nucleic Acids Res. 26:3136‐3145.
   Ho, W.C., Steinbeck, C., and Richert, C. 1999. Solution structure of the aminoacyl‐capped oligodeoxyribonucleotide duplex (W‐TGCGCAC)2 . Biochemistry 38:12597‐12606.
   Juodka, B., Sasnauskiene, S., and Shabarova, Z. 1981. Oligonucleotides and nucleotide‐peptides. 37. On the mechanism of hydrolysis of uridylyl‐(5′ → N)‐amino acids. Intramolecular catalysis by the α‐carboxyl group of amino acids. J. Carbohydr. Nucleosides Nucleotides 8:519‐535.
   Kadkol, S.S., Gage, W.R., and Pasternack, G.R. 1999. In situ hybridization: Theory and practice. Mol. Diagn. 4:169‐183.
   Katritzky, A.R., Munawar, M.A., Kovacs, J., and Khelashvili, L. 2009. Synthesis of amino acid derivatives of quinolone antibiotics. Org. Biomol. Chem. 7:2359‐2362.
   Kibbe, W.A. 2007. OligoCalc: An online oligonucleotide properties calculator. Nucleic Acids Res. 35:43‐46.
   Kottysch, T., Ahlborn, C., Brotzel, F., and Richert, C. 2004. Stabilizing or destabilizing oligodeoxynucleotide duplexes containing single 2′‐deoxyuridine residues with 5‐alkynyl substituents. Chem. Eur. J. 10:4017‐4028.
   Kuyl‐Yeheskiely, E., Tromp, C.M., Lefeber, A.W.M., van der Marel, G.A., and van Boom, J.H. 1988. A convenient approach toward the preparation of nucleopeptides. Tetrahedron 44:6515‐6523.
   Leonova, T.S., Nadeyskaya, E.N., and Yashunskii, V.G. 1987. Synthesis and antibacterial properties of amino acid derivatives of oxolinic acid. Pharm. Chem. J. 21:430‐434.
   Mokhir, A.A., Tetzlaff, C.N., Herzberger, S., Mosbacher, A., and Richert, C. 2001. Monitored selection of DNA‐hybrids forming duplexes with capped terminal C:G base pairs. J. Combin. Chem. 3:374‐386.
   Narayanan, S., Gall, J., and Richert, C. 2004. Clamping down on weak terminal base pairs: Oligonucleotides with molecular caps as fidelity‐enhancing elements at the 5′‐ and 3′‐terminal residues. Nucleic Acids Res. 32:2901‐2911.
   Nimse, S.B., Song, K.‐S., Kim, J., Ta, V.‐T., Nguyen, V.‐T., and Kim, T. 2011. A generalized probe selection method for DNA chips. Chem. Commun. 47:12444‐12446.
   Owczarzy, R., You, Y., Groth, C. L., and Tataurov, A. 2011. Stability and mismatch discrimination of locked nucleic acid‐DNA duplexes. Biochemistry 50:9352‐9367.
   Patra, A. and Richert, C. 2009. High fidelity base pairing at the 3′‐terminus. J. Am. Chem. Soc. 131:12671‐12681.
   Peyret, N., Seneviratne, P.A., Allawi, H.T., and SantaLucia, J. Jr. 1999. Nearest‐neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38:3468‐3477.
   Peyrottes, S., Mestre, B., Burlina, F., and Gait, M.J. 1998. The synthesis of peptide‐oligonucleotide conjugates by a fragment coupling approach. Tetrahedron 54:12513‐12522.
   Pieles, U., Zürcher, W., Schär, M., and Moser, H.E. 1993. Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry: A powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucleic Acids Res. 21:3191‐3196.
   Prestinari, C. and Richert, C. 2011. Intrastrand locks increase duplex stability and base pairing selectivity. Chem. Comm. 47:10824‐10826.
   Pretsch, E., Clerc, T., and Seibl, J. 1986. Tabellen zur Strukturaufklärung organischer Verbindungen: Mit spektroskopischen Methoden. Springer‐Verlag, Berlin.
   Richert, C. 2006. Caps for increased duplex stability and base‐pairing fidelity at termini. Glen Research Report 18:6‐7.
   Richert, C. 2011. A 3′‐Cap for improved target affinity and specificity. Glen Research Report 21:4‐5.
   Richert, C. and Grünefeld, P. 2007. Synthesis and properties of oligonucleotides with acylamido substituents. Synlett 1:1‐18.
   Robles, J., Pedroso, E., and Grandas, A. 1991. Solid‐phase synthesis of a model nucleopeptide with a phosphodiester bond between the 5′ end of a trinucleotide and a serine residue. Tetrahedron Lett. 32:4389‐4392.
   Robles, J., Maseda, M., Beltran, M., Concernau, M., Pedroso, E., and Grandas, A. 1997. Synthesis and enzymatic stability of phosphodiester‐linked peptide‐oligonucleotide hybrids. Bioconjug. Chem. 8:785‐788.
   Saenger, W. 1984. Principles of Nucleic Acid Structure. Springer Verlag, New York.
   Santa Lucia, J. Jr. 1998. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest‐neighbor thermodynamics. Proc. Natl. Acad. Sci. U.S.A. 95:1460‐1465.
   Sarracino, D. and Richert, C. 1996. Quantitative MALDI‐TOF spectrometry of oligonucleotides and a nuclease assay. Bioorg. Med. Chem. Lett. 6:2543‐2548.
   Schena, M., Heller, R.A., Theriault, T.P., Konrad, K., Lachenmeier, E., and Davis, R.W. 1998. Microarrays: Biotechnology's discovery platform for functional genomics. Trends Biotechnol. 16:301‐306.
   Shatkin, A.J. 1976. Capping of eucaryotic mRNAs. Cell 9:645‐653.
   Siegmund, K., Maheshwary, S., Narayanan, S., Connors, W., Riedrich, M., Printz, M., and Richert, C. 2005. Molecular details of quinolone‐DNA interactions: Solution structure of an unusually stable DNA duplex with covalently linked nalidixic acid residues and non‐covalent complexes derived from it. Nucleic Acids Res. 33:4838‐4848.
   Siegmund, K., Ahlborn, C., and Richert, C. 2008. ChipCheckII ‐ Predicting binding curves for multiple analyte strands on small DNA microarrays. Nucleosides Nucleotides Nucleic Acids 27:376‐388.
   Soukchareun, S., Tregear, G.W., and Haralambidis, J. 1995. Preparation and characterization of antisense oligonucleotide peptide hybrids containing viral fusion. Bioconjug. Chem. 6:43‐53.
   Stengele, K.P. and Pfleiderer, W. 1990. Improved synthesis of oligodeoxyribonucleotides. Tetrahedron Lett. 31:2549‐2552.
   Stetsenko, D.A. and Gait, M.J. 2000. Efficient conjugation of peptides to oligonucleotides by “native ligation”. J. Org. Chem. 65:4900‐4908.
   Stetsenko, D.A., Malakhov, A.D., and Gait, M.J. 2002. Total stepwise solid‐phase synthesis of oligonucleotide (3′‐N)‐peptide conjugates. Org. Lett. 4:3259‐3263.
   Tuma, J., Connors, W.H., Stitelman, D.H., and Richert, C. 2002. On the effect of covalently appended quinolones on termini of DNA‐duplexes. J. Am. Chem. Soc. 124:4236‐4246.
   Tuma, J., Paulini, R., Rojas Stütz, J.A., and Richert, C. 2004. How much pi‐stacking do DNA termini seek? Solution structure of a self‐complementary DNA hexamer with trimethoxystilbenes capping the terminal base pairs. Biochemistry 43:15680‐15687.
   Zatsepin, T.S., Stetsenko, D.A., Gait, M.J., and Oretskaya, T.S. 2005. Synthesis of DNA conjugates by solid‐phase fragment condensation via aldehyde‐nucleophile coupling. Tetrahedron Lett. 46:3191‐3195.
   Zhu, Q., Delaney, M.O., and Greenberg, M.M. 2001. Observation and elimination of N‐acetylation of oligonucleotides prepared using fast‐deprotecting phosphoramidites and ultra‐mild deprotection. Bioorg. Med. Chem. Lett. 11:1105‐1107.
Internet Resources
   http://ozone3.chem.wayne.edu/
  Link to HyTher, a program predicting melting points of oligonucleotide duplexes.
   http://www.basic.northwestern.edu/biotools/oligocalc.html
  For calculations of oligonucleotide extinction coefficients.
  http://omlc.ogi.edu/spectra/PhotochemCAD/index.html
  Database for extinction coefficients of small molecules.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library