Preparation of Oligodeoxyribonucleotides Containing the Pyrimidine(6–4)pyrimidone Photoproduct by Using a Dinucleotide Building Block
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
This unit describes procedures for the synthesis of a dinucleotide?type building block of the pyrimidine(6?4)pyrimidone photoproduct [(6?4) photoproduct], which is one of the major DNA lesions induced by ultraviolet (UV) light, and its incorporation into oligodeoxyribonucleotides. Although this type of lesion is frequently found at thymine?cytosine sites, the building block of the (6?4) photoproduct formed at thymine?thymine sites can be synthesized much more easily. The problem in the oligonucleotide synthesis is that the (6?4) photoproduct is labile under alkaline conditions. Therefore, building blocks with an amino?protecting group that can be removed by a brief treatment with ammonia water at room temperature must be used for the incorporation of the normal bases. Byproduct formation by the coupling of phosphoramidites with the N3 of the 5? component should also be considered. This side reaction can be avoided by using benzimidazolium triflate as an activator. Curr. Protoc. Nucleic Acid Chem . 53:4.56.1?4.56.18. © 2013 by John Wiley & Sons, Inc.
Keywords: oligonucleotides; UV damage; DNA repair; xeroderma pigmentosum
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Synthesis of Oligonucleotides Containing the (6–4) Photoproduct
- Alternate Protocol 1: Synthesis of Oligonucleotides Using Tetrazole as an Activator
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1:
Materials
Basic Protocol 2:
Materials
Basic Protocol 3:
Materials
Basic Protocol 4: Synthesis of Oligonucleotides Containing the (6–4) Photoproduct
Materials
Alternate Protocol 1: Synthesis of Oligonucleotides Using Tetrazole as an Activator
Materials
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Figure 4.56.1 Synthesis of 8 . Abbreviations: DMT, 4,4′‐dimethoxytrityl; DCC, 1,3‐dicyclohexylcarbodiimide; DMAP, 4‐(dimethylamino)pyridine. View Image -
Figure 4.56.2 (A ) HPLC analyses before (i) and after (ii and iii) the UV irradiation of 4 . The monitored wavelengths were 254 (i and ii) and 325 nm (iii), and the conditions are described in the text (see , step 11). (B and C ) UV absorption spectra of 4 (B) and 5 (C). View Image -
Figure 4.56.3 HPLC analyses of crude samples of a 33‐mer, d(CTCGTCAGCATC‐T(6–4)T‐CATCATACAGTCAGTGGCA), synthesized by using BIT (A ) and tetrazole (B ). The monitored wavelengths were 254 (i) and 325 nm (ii), and in each panel, trace ii is enlarged 10‐fold, in comparison to trace i. The conditions are described in the text (see , step 13). From the UV absorption spectrum, the peak with the retention time of 9.1 min was assigned to the protecting group released from the oligonucleotide, which was not removed by extraction. View Image
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Literature Cited
| Literature Cited | |
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