Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

The preparation of two reversible biotinylation phosphoramidites and their application in labeling and affinity purification of synthetic oligodeoxyribonucleotides will be described. In both cases, the biotin is linked to the 5??terminus of DNA through a diisopropyl silyl acetal functionality. This linkage is completely stable under certain postsynthetic cleavage/deprotection conditions, but can be readily broken by fluoride ions, releasing unmodified 5??OH and 5??phosphate DNA, respectively. To demonstrate the use of these reversible biotinylation methods, crude DNA was incubated with NeutrAvidin?coated microspheres, full?length biotinylated DNA was efficiently attached to the solid phase, and nonbiotinylated failure sequences and other impurities were readily removed by washing with buffer. Cleavage of the silyl acetal linkage afforded high?quality, full?length, unmodified 5??OH and 5??phosphate DNA, respectively, depending on which of the two phosphoramidites was used. It is anticipated that this method will find applications in areas that require efficient isolation of DNA from a complex mixture.

Keywords: DNA; Phosphoramidite; Biotinylation; Fluoride Cleavage; Silyl Acetal; Avidin; Affinity Purification

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Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA
Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA
Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres
Support Protocol 2: Synthesis of Biotinyl Alcohol
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA

Materials

Biotinyl alcohol S.1 (see protocol 4 for preparation)
Imidazole (99%)
Argon and nitrogen gas
N ,N ‐Dimethylformamide (DMF, anhydrous)
Diisopropylethylamine (DIEA)
Diisopropyldichlorosilane (Fluka)
Thymidine (99%+)
Ethyl acetate
5% (w/v) sodium bicarbonate (NaHCO₃ )
Sodium sulfate (Na₂ SO₄ , anhydrous)
Chloroform (CHCl₃ )
Methanol
40‐µm silica gel (Baker)
TLC plates: silica gel on aluminum (60F‐254, 200‐µm thickness)
Methylene chloride (CH₂ Cl₂ , anhydrous)
2‐Cyanoethyl‐N ,N ,N ′,N′‐tetraisopropylphosphoramidite (2‐cyanoethyl‐ tetraisopropylphosphorodiamidite, 97%)
1H ‐Tetrazole (99%+, sublimed)
Tetrahydrofuran (THF, 99%+)
5′‐DMTr, 2‐cyanoethyl phosphoramidite monomers:
- 5′‐O ‐dimethoxytrityl‐N ⁶ ‐benzoyl‐2′‐deoxyadenosine‐3′‐(2‐cyanoethyl‐N ,N ‐di‐isopropyl)phosphoramidite (dA^Bz )
- 5′‐O ‐dimethoxytrityl‐N ² ‐isobutyryl‐2′‐deoxyguanosine‐3′‐(2‐cyanoethyl‐N ,N ‐di‐isopropyl)phosphoramidite (dG^{i ‐Bu} )
- 5′‐O ‐dimethoxytrityl‐N ⁴ ‐acetyl‐2′‐deoxycytidine‐3′‐(2‐cyanoethyl‐N ,N ‐diisopro‐pyl)phosphoramidite (dC^Ac )
- 5′‐O ‐dimethoxytrityl‐2′‐deoxythymidine‐3′‐(2‐cyanoethyl‐N ,N ‐diisopropyl)phos‐phoramidite (T)

Acetonitrile (CH₃ CN, anhydrous)
∼29% (v/v) ammonium hydroxide (NH₄ OH)
∼40% (v/v) methylamine (CH₃ NH₂ )

50‐mL one‐neck round‐bottomed flasks (oven dried)
Vacuum pump
10‐, 5‐, 1‐, 0.25‐mL graduated glass syringes
20‐G, 5‐cm stainless steel needles
Septa (14/20, 24/40)
250‐mL separatory funnel
Filter funnel and Whatman no. 1 filter paper
Rotary evaporator equipped with a water aspirator
Flash chromatography columns (4.5 × 12–cm, 3.0 × 12–cm)
UV lamp
5‐mL screw‐cap vials
Speedvac evaporator

Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D ), column chromatography ( appendix 3E ), solid‐phase automatic DNA synthesis ( appendix 3C ) using classical phosphoramidite method (unit 3.3 ), and purification of oligonucleotides by SDS‐PAGE (unit 10.4 ) and reversed‐phase HPLC (unit 10.5 )

Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA

Materials

Biotinyl alcohol S.1 (see protocol 4 for preparation)
Imidazole (99%)
Argon and nitrogen gas
N,N ‐Dimethylformamide (DMF, anhydrous)
Diisopropylethylamine (DIEA)
Diisopropyldichlorosilane (Fluka)
Diethyl bis(hydroxymethyl)malonate (97%)
Methylene chloride (CH₂ Cl₂ )
5% (v/v) citric acid
Sodium sulfate (Na₂ SO₄ , anhydrous)
Chloroform (CHCl₃ )
Methanol
40‐µm silica gel (Baker)
TLC plates: silica gel on aluminum (60F‐254, 200‐µm thickness)
Acetonitrile (distilled over CaH₂ )
2‐Cyanoethyl‐N ,N ,N ′,N′‐tetraisopropylphosphoramidite (2‐cyanoethyl‐ tetraisopropylphosphorodiamidite, 97%)
0.45 M 1H ‐tetrazole (99%+, sublimed) in acetonitrile
5% (w/v) sodium bicarbonate (NaHCO₃ )
Tetrahydrofuran (THF, 99%+)
Triethylamine (TEA)

50‐mL one‐neck, round‐bottomed flasks (oven dried)
Vacuum pump
20‐G, 5‐cm stainless steel needles
5‐, 1‐, and 0.5‐mL graduated glass syringes
Septa (14/20, 24/40)
250‐mL separatory funnels
Filter funnel and Whatman no. 1 filter paper
Rotary evaporator equipped with a water aspirator
Flash chromatography columns (3.0 × 12–cm, 3.0 × 10–cm)
UV lamp

Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D ), column chromatography ( appendix 3E ), and incorporation of biotinylated phosphoramidite into oligonucleotide (see protocol 1 )

Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres

Materials

UltraLink Immobilized NeutrAvidin (1000‐Å pore size; 50‐ to 80‐µm particle size; 0.08 µmol biotin/ml gel binding capacity; Pierce), prepared as 50% (v/v) slurry containing 0.02% (w/v) sodium azide
PBS ( appendix 2A )
Biotinylated DNA (see protocol 1 for S.4 or protocol 2 for S.8 )
Acetone (dried over anhydrous sodium sulfate)
Tetrahydrofuran (THF, distilled over sodium/benzophenone ketyl)
70:30 (v/v) hydrogen fluoride in pyridine (HF/pyridine)
Methoxytrimethylsilane
∼40% (v/v) methylamine (CH₃ NH₂ ; for 5′‐phosphate DNA S.8 only)
10‐ and 1.5‐mL centrifuge tubes
Lyophilizer or Speedvac evaporator

Support Protocol 2: Synthesis of Biotinyl Alcohol

Levulinic acid (98%)
Dry ice/acetone
3.0 M methyl magnesium bromide in ether
Acetic acid (99.7%+)
Magnesium sulfate (MgSO₄ , anhydrous)
2,2′‐(Ethylenedioxy)bis(ethylamine) (98%)
Ether
Triethylamine
Ninhydrin
Ethanol (anhydrous)
Biotin (AnaSpec)
4‐(Dimethylamino)pyridine (DMAP)
Pyridine (distilled from CaH₂ )
t ‐Butylchlorodiphenylsilane (98%)
4‐t ‐Butylbenzoylchloride (98%)
Potassium carbonate (K₂ CO₃ )
5% citric acid
Isobutylchloroformate (98%)

100‐, 20‐, 10‐, 5‐, and 1‐mL graduated glass syringes
1000‐mL two‐neck round‐bottomed flask, oven dried
Condenser
500‐ and 250‐mL separatory funnels
Aldrich short‐path distillation apparatus
50‐, 100‐ and 250‐mL one‐neck round‐bottomed flasks, oven dried
Flash chromatography columns (4.5 × 30–cm and 4.5 × 10–cm)
Heat gun
Rubber septum
2‐ and 6‐cm stainless steel needles

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Figures

Figure 4.20.1 Preparation of reversible biotinylation phosphoramidite S.3 and structures of biotinylated 5′‐OH DNAs S.4 and S.5 .

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Figure 4.20.2 HPLC profiles of DNAs S.5 (trace a), 5′‐TACAGTGACT‐3′ (trace b), S.9 (trace c), and 5′‐H₂ O₃ PO‐ACAGTGACT‐3′ (trace d) generated on a C18 reversed‐phase column (100 Å, 250 × 4.6 mm, Varian Analytical Instruments), using a linear gradient of 0% to 45% solvent B (90% acetonitrile) in solvent A (0.1 M triethylammonium acetate, 5% acetonitrile) over 60 min at a flow rate of 1 mL/min by detecting the absorbance of DNA at 260 nm. For a detailed protocol for performing reversed‐phase HPLC, refer to UNIT .

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Figure 4.20.3 Preparation of reversible biotinylation phosphoramidite S.7 and structures of biotinylated 5′‐phosphate DNAs S.8 and S.9 .

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Figure 4.20.4 Removal of biotin from biotinylated DNAs S.4 and S.8 to generate unmodified 5′‐OH DNA S.10 and unmodified 5′‐phosphate DNA S.12 , respectively.

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Figure 4.20.5 Preparation of the biotinyl alcohol S.1 .

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Videos

Literature Cited

Literature Cited
	Agrawal, S., Christodoulou, C., and Gait, M.J. 1986. Efficient methods for attaching non‐radioactive labels to the 5′ ends of synthetic oligodeoxyribonucleotides. Nucl. Acids Res. 14:6227‐6245.
	Cadet, J. and Vigny, P. 1990. The photochemistry of nucleic acids. In Bioorganic Photochemistry (H. Morrison, ed.) Vol. 1, pp. 170‐184. John Wiley & Sons, New York.
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	De Vos, M.J., Van Elsen, A., and Bollen, A. 1994. New non‐nucleosidic phosphoramidites for the solid phase multi‐labeling of oligonucleotides: Comb‐ and multifork‐like structure. Nucleosides Nucleotides 13:2245‐2265.
	Fang, S. and Bergstrom, D.E. 2003a. Reversible biotinylation phosphoramidite for 5′‐end‐labeling, phosphorylation and affinity purification of synthetic oligonucleotides. Bioconjugate Chem. 14:80‐85.
	Fang, S. and Bergstrom, D.E. 2003b. Fluoride‐cleavable biotinylation phosphoramidite for 5′‐end‐labeling, affinity purification of synthetic oligonucleotides. Nucl. Acids Res. 31:708‐715.
	Gildea, B.D., Coull, J.M., and Koster, H. 1990. A versatile acid‐labile linker for modification of synthetic biomolecules. Tetrahedron Lett. 31:7095‐7098.
	Greenberg, M.M. 1995. Photochemical release of protected oligodeoxyribonucleotides containing 3′‐glycolate termini. Tetrahedron 51:29‐38.
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	Langer, P.R., Waldrop, A.A., and Ward, D.C. 1981. Enzymatic synthesis of biotin‐labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78:6633‐6637.
	McInnes, J.L. and Symons, R.H. 1989. Preparation and detection of nonradioactive nucleic acid and oligonucleotide probes. In Nucleic Acid Probes (R.H. Symons, ed.) pp. 33‐80. CRC Press, Boca Raton, Fla.
	Neuner, P. 1996. New non nucleosidic phosphoramidite reagent for solid phase synthesis of biotinylated oligonucleotides. Bioorg. Med. Chem. Lett. 6:147‐152.
	Olejnik, J., Krzymanska‐Olejnik, E., and Rothschild, K.J. 1996. Photocleavable biotin phosphoramidite for 5′‐end‐labelling, affinity purification and phosphorylation of synthetic oligonucleotides. Nucl. Acids Res. 24:361‐366.
	Pon, R.T. 1991. A long chain biotin phosphoramidite reagent for the automated synthesis of 5′‐biotinylated oligonucleotides. Tetrahedron Lett. 32:1715‐1718.
	Reddy, M.P., Hanna, N.B., and Farooqui, F. 1994. Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 35:4311‐4314.
	Shimkus, M., Levy, J., and Herman, T. 1985. A chemically cleavable biotinylated nucleotide: Usefulness in the recovery of protein‐DNA complexes from avidin affinity columns. Proc. Natl. Acad. Sci. U.S.A. 82:2593‐2597.
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Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification

Abstract

Table of Contents

Materials

Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA

Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA

Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres

Support Protocol 2: Synthesis of Biotinyl Alcohol

Figures

Videos

Literature Cited