5′‐Iodination of Solid‐Phase‐Linked Oligodeoxyribonucleotides

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

5??Iodinated oligodeoxyribonucleotides readily react with 3??phosphorothioated DNA in the presence of a complementary template to yield a conjugate that is identical to natural DNA in every respect except that one oxygen atom in the phosphodiester backbone is replaced by a sulfur atom. The 5??iodo group is easily converted to a variety of other functional groups and will quickly react with thiol?containing labels to yield stable thioether conjugates. This unit presents manual and automated procedures for converting the 5??hydroxyl of protected CPG?bound oligodeoxyribonucleotides to an iodo group and for releasing and purifying the products.

Keywords: iodination; 5??modification; solid?phase; oligodeoxyribonucleotide; electrophile; ligation

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Basic Protocol 1: Manual Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support
Alternate Protocol 1: Automated Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Manual Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support

Materials

Anhydrous N ,N ‐dimethylformamide (DMF)
Iodination solution (see recipe )
Methylene chloride (CH₂ Cl₂ )
28% (v/v) ammonium hydroxide (NH₄ OH)

DNA synthesizer (e.g., ABI; see appendix 3C )
0.2 to 10 µmol DNA synthesis column with long‐chain alkylamine controlled‐pore glass (CPG) support
1‐ and 10‐mL syringes
Shaker, rocker, or other agitating device
C18 reversed‐phase HPLC column (unit 10.5 )
Speedvac evaporator

Additional reagents and equipment for oligonucleotide synthesis (Chapters 3 and 4 and appendix 3C ) and reversed‐phase HPLC (unit 10.5 )

Alternate Protocol 1: Automated Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support

Glass wool or line filter (e.g., preparation and delivery line filter from ABI)
Empty DNA synthesis column (e.g., 1000‐Å CPG column; ABI)
10‐mL syringes
Clean, oven‐dried phosphoramidite bottle compatible with synthesizer
Reagent bottle compatible with synthesizer, filled with anhydrous DMF

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Figures

Figure 4.19.1 Iodination of solid‐phase‐linked oligodeoxyribonucleotides. Abbreviations: B₁ and B₂ , thymine and/or N ‐protected nucleobases; CPG, long‐chain alkylamine controlled‐pore glass (resin).

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Figure 4.19.2 RP‐HPLC profiles of 5′‐d(NGTAGGCAAGAGT) before and after 5′‐iodination. From top to bottom, N = T, C, A, and G, respectively. Reprinted from Miller and Kool () with permission from the American Chemical Society.

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Literature Cited

	Dimitrijevich, S.D., Verheyden, J.P.H., and Moffatt, J.G. 1979. Halo sugar nucleosides. 6. Synthesis of some 5′‐deoxy‐5′‐iodo and 4′,5′‐unsaturated purine nucleosides. J. Org. Chem. 44:400‐406.
	Miller, G.P. and Kool, E.T. 2002. A simple method for electrophilic functionalization of DNA. Org. Lett. 4:3599‐3601.
	Xu, Y. and Kool, E.T. 1997. A novel 5′‐iodonucleoside allows efficient non‐enzymatic ligation of single‐stranded and duplex DNAs. Tetrahedron Lett. 38:5595‐5598.
	Xu, Y. and Kool, E.T. 1998. Chemical and enzymatic properties of bridging 5′‐S‐phosphorothioester linkages in DNA. Nucl. Acids Res. 26:3159‐3164.
Key References
	Miller and Kool, 2002. See above.
	Reports a method to iodinate the 5′‐carbon of oligo‐deoxyribonucleotides on a solid support.