Preparation of α‐Oxo Semicarbazone Oligonucleotide Microarrays

Oleg Melnyk1, Christophe Olivier1, Nathalie Ollivier1, Yves Lemoine2, David Hot2, Ludovic Huot2, Catherine Gouyette3

1 Université de Lille 2, Lille, 2 Institut Pasteur de Lille, Lille, 3 Institut Pasteur, Paris
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.6
DOI:  10.1002/0471142700.nc1206s19
Online Posting Date:  January, 2005
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Abstract

This unit describes the preparation of α‐oxo aldehyde functionalized oligodeoxynucleotides, the preparation and characterization of semicarbazide glass slides, and the fabrication of α‐oxo semicarbazone microarrays by site‐specific ligation of α‐oxo‐aldehyde oligodeoxynucleotides to the semicarbazide glass slides. The α‐oxo aldehyde group COCHO is extensively used in ligation chemistry for the preparation of large molecular constructs. It is stable toward air oxidation and mainly present in aqueous solution in the hydrated form COC(OH)2. It reacts efficiently with hydrazine derivatives, in particular, with the semicarbazide group. The reaction occurs spontaneously in water at pH 5.5. Site‐specific immobilization of glyoxylyl oligodeoxynucleotides on semicarbazide glass slides allows the preparation of high‐quality microarrays that can be used directly in hybridization experiments.

Keywords: microarray; oligonucleotide; α‐oxo semicarbazone; α‐oxo aldehyde; semicarbazide

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis and Characterization of Glyoxylyl Oligodeoxynucleotides
  • Support Protocol 1: Preparation of ODNs for MALDI‐TOF‐MS
  • Basic Protocol 2: Preparation and Characterization of Semicarbazide‐Functionalized Glass Slides
  • Support Protocol 2: Synthesis of Peptide 1: Rho‐Lys‐Arg‐NH‐(CH2)3‐NH‐COCHO
  • Support Protocol 3: Synthesis of Peptide 2: Rho‐Lys‐Arg‐NH2
  • Basic Protocol 3: Preparation of α‐Oxo Semicarbazone ODN Microarrays
  • Support Protocol 4: Quantitation of ODN‐COCHO Immobilized onto Semicarbazide Glass Slides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis and Characterization of Glyoxylyl Oligodeoxynucleotides

  Materials
  • 18‐(O‐Dimethoxytrityl)hexaethyleneglycol‐1‐[(2‐cyanoethyl)‐(N,N‐diisopropyl)]phosphoramidite (Glen Research)
  • 6‐(4‐Monomethoxytritylamino)hexyl‐(2‐cyanoethyl)‐(N,N‐diisopropyl)phosphoramidite (Glen Research)
  • 3% (v/v) trichloroacetic acid in dichloromethane (TCA/DCM)
  • Acetonitrile
  • Argon
  • Compressed air
  • 2,6‐Lutidine
  • Anhydrous tetrahydrofuran (THF)
  • (+)‐Diacetyl‐L‐tartaric anhydride (Aldrich)
  • 32% concentrated ammonia solution
  • HPLC buffer A: 99:1 (v/v) 10 mM tetraethylamine acetate (TEAA), pH 6.5, in acetonitrile
  • HPLC buffer B: 95:5 (v/v) acetonitrile/deionized water
  • 3‐Hydroxypicolinic acid (3‐HPA) matrix
  • 100 mM sodium phosphate buffer, pH 6.6
  • 1 mM sodium periodate in deionized water
  • Tartaric acid
  • Tri‐n‐butylphosphine
  • D‐Mannitol
  • Synthesis column with DMTr‐protected controlled‐pore glass (CPG) support (1‐µmol scale)
  • 1‐mL glass syringes
  • 1.5‐mL screw‐cap vials
  • 55°C oven
  • UV spectrophotometer and cuvettes
  • 5‐mL flask
  • Rotary evaporator
  • 250 × 4.6–mm and 300 × 12.5–mm C18 RP‐HPLC columns
  • Lyophilizer
  • 1.5‐mL microcentrifuge tube
  • Additional reagents and equipment for automated DNA synthesis (unit 3.3, appendix 3C), RP‐HPLC (unit 10.5), MALDI‐TOF‐MS (see protocol 2 and unit 10.1), and quantitation of DNA (unit 10.3)

Support Protocol 1: Preparation of ODNs for MALDI‐TOF‐MS

  Materials
  • Dowex 1×8 H+ (100 to 200 mesh) cation‐exchange resin
  • 1 M ammonium acetate
  • Acetone
  • n‐Hexane
  • 3‐Hydroxypicolinic acid (3‐HPA)
  • ODN sample(s) to be analyzed (see protocol 1)
  • 0.1 M diammonium citrate
  • 250‐mL Erlenmeyer flask
  • 500‐mL filtration flask
  • 70‐mm‐diameter Buchner funnel
  • Vacuum pump
  • Parafilm
  • 0.5‐mL microcentrifuge tubes
  • MALDI‐TOF mass spectrometer (PE Biosystems)

Basic Protocol 2: Preparation and Characterization of Semicarbazide‐Functionalized Glass Slides

  Materials
  • 25% hydrazine hydrate in water
  • Fluorenylmethylchloroformate (Fmoc‐Cl)
  • Acetonitrile
  • Absolute ethanol
  • Phosphorus pentoxide (P 2O 5)
  • 35% (v/v) hydrogen peroxide (H 2O 2) in water
  • Sulfuric acid (H 2SO 4)
  • Methanol
  • 3‐Aminopropyltrimethoxysilane
  • Triphosgene
  • 1,2‐Dichloroethane
  • Diisopropylethylamine (DIPEA)
  • N,N‐Dimethylformamide (DMF)
  • Piperidine
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐en (DBU)
  • Formamide
  • Diiodomethane
  • Peptide 1: Rho‐Lys‐Arg‐NH‐(CH 2) 3‐NH‐COCHO (see protocol 4)
  • Peptide 2: Rho‐Lys‐Arg‐CONH 2 (see protocol 5)
  • 100 mM sodium acetate buffer, pH 5.5
  • 5% aqueous K 2HPO 4
  • 0.1 M tris(hydroxymethyl)aminomethane acetate (Tris acetate), pH 5.5, containing 0.1% (v/v) Tween 20
  • 2‐L flask with a 500‐mL dropping funnel
  • Rotary evaporator equipped with a vacuum pump
  • Water condenser
  • Oil bath
  • 1‐L filter flask with a filter adapter and fritted glass
  • Glass microscope slides (e.g., 75 × 25–mm, frosted, ESCO Precleaned Micro Slide)
  • Glass staining dishes for 20 slides, with covers, slide racks, and slide rack handle (e.g., Wheaton)
  • 750‐mL Teflon PFA dish (15 × 10 × 5–cm, e.g., Bioblock)
  • 1‐L Teflon PFA beaker with handle (e.g., Nalgene Labware)
  • Teflon tweezers
  • 500‐ and 1000‐mL Erlenmeyer flasks
  • Sonicator, 40 kHz (e.g., Branson)
  • 110°C drying oven
  • Desiccator
  • Vacuum pump
  • Dust‐proof container for slides
  • Goniometer
  • Microarray scanner with Cy3 channel
  • Fluorescence analysis software

Support Protocol 2: Synthesis of Peptide 1: Rho‐Lys‐Arg‐NH‐(CH2)3‐NH‐COCHO

  Materials
  • Novasyn TG resin (Novabiochem)
  • SurfaSil siliconizing fluid (Interchim)
  • Dichloromethane (DCM)
  • N,N‐Dimethylformamide (DMF)
  • Fmoc‐Val‐OH
  • 2‐(1H‐Benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU)
  • N‐Hydroxybenzotriazole (HOBT)
  • Diisopropylethylamine (DIPEA)
  • 2,4,6‐Trinitrobenzenesulfonic acid (TNBS)
  • 20% piperidine in DMF
  • Dimethyl‐2,3‐O‐isopropylidene‐L‐tartrate (Aldrich)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU, 97%, Aldrich)
  • 4‐Dimethylaminopyridine (DMAP)
  • Benzotriazole‐1‐yl‐oxy‐tris‐pyrrolidinophosphonium hexafluorophosphate (PyBOP)
  • 1,3‐Diaminopropane
  • Fmoc‐Arg(Pbf)‐OH
  • Fmoc‐Lys(Boc)‐OH
  • 5(6)‐Carboxytetramethylrhodamine (Rho)
  • Trifluoroacetic acid (TFA)
  • Thioanisole
  • Phenol
  • 1,2‐Ethanedithiol
  • Triisopropylsilane (TIS)
  • 33% acetic acid (AcOH)
  • NaIO 4
  • Ethanolamine
  • Acetonitrile
  • Eluent A: water containing 0.05% (v/v) TFA
  • Eluent B: water/25% (v/v) acetonitrile containing 0.05% TFA
  • 60‐mL glass manual solid‐phase peptide synthesis reactor, closed with an inert screw cap at the top and a sealed‐in fritted glass at the bottom, connected by a lower stopcock (customized by Vasse Industries)
  • Vacuum flask and Teflon vacuum pump
  • Automatic shaker
  • Analytical and preparative HPLC systems with 300 × 12.5–mm and 250 × 4.6–mm C18 columns
  • Lyophilizer
  • Vacuum pump
  • Additional reagents and equipment for TNBS test (Chan and White, ), RP‐HPLC (unit 10.5), and mass spectrometry

Support Protocol 3: Synthesis of Peptide 2: Rho‐Lys‐Arg‐NH2

  • Novasyn TGR resin (Novabiochem)
  • 1:1 (v/v) diethyl ether/n‐heptane

Basic Protocol 3: Preparation of α‐Oxo Semicarbazone ODN Microarrays

  Materials
  • ODN‐COCHO (glyoxylyl ODN; see protocol 1)
  • 20× SSC, pH 7.0 ( appendix 2A)
  • 1 N HCl
  • Saturated NaCl
  • 0.2% (w/v) SDS
  • Speedvac evaporator (Savant)
  • 96‐well microtiter plates, V‐shaped, low profile (ABGene)
  • Semicarbazide glass slides (see protocol 3)
  • Arrayer (e.g., Affymetrix 427 Arrayer)
  • Metallic slide rack
  • Hermetic plastic box
  • Temperature‐controlled incubator

Support Protocol 4: Quantitation of ODN‐COCHO Immobilized onto Semicarbazide Glass Slides

  • 32P]ddATP (Amersham Biosciences)
  • 5× terminal transferase reaction buffer: 1 M potassium cacodylate, 125 mM Tris⋅Cl, 1.25 mg/mL bovine serum albumin, pH 6.6 at 25°C
  • Terminal transferase
  • Qiaquick nucleotide removal kit (Qiagen) which includes:
    • PN solution
    • Qiaquick nucleotide removal column
    • PE buffer
  • DNase‐ and RNase‐free distilled water
  • 1.5‐ and 2‐mL microcentrifuge tubes
  • 37°C water bath
  • Benchtop centrifuge
  • Scintillation counter
  • Vacuum desiccator
  • Plastic film (e.g., Saran Wrap)
  • Phosphorimager (e.g., Cyclone Storage Phosphor System, Packard BioScience)
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Figures

Videos

Literature Cited

Literature Cited
   Balladur, V., Theretz, A., and Mandrand, B. 1997. Determination of the main forces driving DNA oligonucleotide adsorption onto aminated silica wafers. J. Colloid Interface Sci. 194:408‐418.
   Chan, W.C. and White, P.D. 1999. Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W.C. Chan and P.D. White, eds.) pp. 61‐62. Oxford University Press, Oxford.
   DeRisi, J., Penland, L., Brown, P.O., Bittner, M.L., Meltzer, P.S., Ray, M., Chen, Y., Su, Y.A., and Trent, J.M. 1996. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat. Genet. 14:457‐460.
   Duburcq, X., Olivier, C., Desmet, R., Halasa, M., Carion, O., Grandidier, B., Heim, T., Stievenard, D., Auriault, C., and Melnyk, O. 2004. Polypeptide semicarbazide glass slide microarrays: Characterization and comparison with amine slides in serodetection studies. Bioconjug. Chem. 15:317‐325.
   Fields, G.B. and Noble, R.L. 1990. Solid phase peptide synthesis utilizing 9‐fluorenyl‐methoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161‐214.
   Gait, M.J. 1984. An introduction to modern methods of DNA synthesis. In Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed.) pp. 1‐22. IRL Press, Oxford.
   Kwok, D.Y. and Neumann, A.W. 1999. Contact angle measurement and contact angle interpretation. Adv. Colloid Interface Sci. 81:167‐249.
   Melnyk, O., Fruchart, J.S., Grandjean, C., and Gras‐Masse, H. 2001. Tartric acid‐based linker for the solid‐phase synthesis of C‐terminal peptide α‐oxo aldehydes. J. Org. Chem. 66:4153‐4160.
   Olivier, C., Hot, D., Huot, L., Ollivier, N., El‐Madhi, O., Gouyette, C., Huynh‐Dinh, T., Gras‐Masse, H., Lemoine, Y., and Melnyk, O. 2003. α‐Oxo semicarbazone peptide or oligodeoxynucleotide microarrays. Bioconjug. Chem. 14:430‐439.
   Podyminogin, M.A., Lukhtanov, E.A., and Reed, M. 2001. Attachment of benzaldehyde‐modified oligodeoxynucleotide probes to semicarbazide‐coated glass. Nucl. Acids Res. 29:5090‐5098.
   Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467‐470.
   Zhang, R.E., Cao, Y.L., and Hearn, M.W. 1991. Synthesis and application of Fmoc‐hydrazine for the quantitative determination of saccharides by reversed‐phase high‐performance liquid chromatography in the low and subpicomole range. Anal. Biochem. 195:160‐167.
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