Oligodeoxynucleotide Containing S‐Functionalized 2′‐Deoxy‐6‐Thioguanosine: Facile Tools for Base‐Selective and Site‐Specific Internal Modification of
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Abstract
Chemically modified oligonucleotides play a significant role for genomic research. Modified nucleosides, such as with a fluorescent dye, can be obtained by chemical synthesis. Site?specifically modified long nucleic acids are obtained by ligation of chemically modified short oligonucleotides with enzyme, photochemistry, or catalytic DNA. The functionality?transfer ODN (FT?ODN), which contains 2??deoxy?6?thioguanosine (6?thio?dG) functionalized with the 2?methyliden?1,3?diketone group, is hybridized with the target RNA to trigger the selective functionalization of the 4?amino group of the cytosine base at pH 7 or the 2?amino group of the guanine base at pH 9.4 or at pH 7.4 in the presence of NiCl2 . In particular, the functionality?transfer reaction (FTR) under the alkaline conditions or neutral conditions in the presence of NiCl2 proceeds rapidly and selectively to lead to the modification of the target guanine. The transfer reaction of the acetylene?containing diketone group produces the acetylene?modified RNA, which can be subjected to the Cu(I)?catalyzed ?click chemistry? with a variety of azide compounds for highly specific, internal modification of RNA. Curr. Protoc. Nucleic Acid Chem. 48:4.49.1?4.49.16. © 2012 by John Wiley & Sons, Inc.
Keywords: 2??deoxy?6?thioguanosine; RNA modification; functionality transfer; click chemistry
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Preparation of Dimethoxytrityl‐Protected Phophoramidite of S‐Protected 2′‐Deoxy‐6‐Thioguanosine from 2′‐Deoxyguanosine
- Basic Protocol 2: Preparation of 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione
- Basic Protocol 3: Preparation of 2′‐Deoxy‐6‐Thioguanosine Containing Oligodeoxynucleotides and Its Functionalization with 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1: Preparation of Dimethoxytrityl‐Protected Phophoramidite of S‐Protected 2′‐Deoxy‐6‐Thioguanosine from 2′‐Deoxyguanosine
Materials
Basic Protocol 2: Preparation of 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione
Materials
Basic Protocol 3: Preparation of 2′‐Deoxy‐6‐Thioguanosine Containing Oligodeoxynucleotides and Its Functionalization with 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione
Materials
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Figure 4.49.1 The nitrosyl group transfer from 2′‐deoxy‐6‐thioguanosine (S.1 ) to the 4‐amino group of cytosine (S.3 ). View Image -
Figure 4.49.2 The functionality transfer reaction of 1,3‐diketone derivative from 2′‐deoxy‐6‐thioguanosine (S.6 ) to the 4‐amino group of cytosine (S.8 ) at pH 7.0. The transfer reaction takes place selectively to the 2‐amino group of guanine (S.9 ) at pH 9.6 or at pH 7.4 in the presence of NiCl2 . View Image -
Figure 4.49.3 Functionality transfer reaction leading to the internal RNA modification. The acetylene group of the modified RNA (S.11 ) is useful as a scaffold for labeling with a variety of molecules (S.12 ) through the “click chemistry” View Image -
Figure 4.49.4 Odorless synthesis of the 2′‐deoxy‐6‐thioguanosine derivative and its phosphoroamidite (S.15 ). View Image -
Figure 4.49.5 The synthesis of the transfer units S.17 and S.19 . View Image -
Figure 4.49.6 Preparation of the functionalized ODN2 and change of HPLC profile. View Image -
Figure 4.49.7 Functionality transfer reaction by ODN2 to modify 4‐amino group of the target cytosine at pH 7.0 or to modify 2‐amino group of the target guanine at pH 9.4. Time course of the yields of RNA4a (on the hour time‐scale) and RNA6b (on the minute time‐scale) were obtained by following the reaction by HPLC. View Image -
Figure 4.49.8 Cu(I)‐catalyzed “click chemistry” between the modified RNA and the azide compound produces the internally modified RNA7 . The HPLC chart represents the change of the reaction with the azide derivative of biotin. View Image
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Literature Cited
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