Site directed nitroxide spin labeling of oligonucleotides for NMR and EPR studies

被引：9

作者：

Shepherd, Nicholas E. ^{[1
]}

Gamsjaegera, Roland ^{[1
,2
]}

Vandevenne, Marylene ^{[1
]}

Cubeddua, Liza ^{[1
,2
]}

Mackay, Joel P. ^{[1
]}

机构：

[1] Univ Sydney, Sch Mol Biosci, Sydney, NSW 2006, Australia

[2] Univ Western Sydney, Sch Sci & Hlth, Penrith, NSW 2751, Australia

来源：

TETRAHEDRON | 2015年 / 71卷 / 05期

基金：

澳大利亚研究理事会;

关键词：

Nitroxide; Phosphorothioate; Nuclear magnetic resonance spectroscopy; Electron paramagnetic resonance spectroscopy; Paramagnetic relaxation enhancement; PARAMAGNETIC RELAXATION ENHANCEMENT; NUCLEIC-ACIDS; SPECTROSCOPY; DNA; RNA; BINDING;

D O I：

10.1016/j.tet.2014.12.056

中图分类号：

O62 [有机化学];

学科分类号：

070303 ; 081704 ;

摘要：

Nitroxide labels are useful probes of biomolecule structure that can be detected by NMR and EPR spectroscopy. Although many methods exist for labeling oligonucleotides with nitroxides, most require reagents that are expensive or laborious to prepare. A simpler approach is described herein using commercially available phosphorothioate oligonucleotides and 2-(3-iodoacetamidomethyl)-PROXYL. We describe semi-optimized conditions for labeling DNA and RNA oligonucleotides and methodology for purifying and identifying these reagents by MALDI-MS. The nitroxide label showed some propensity to hydrolyze at high temperature and over prolonged periods at room temperature. Nitroxide-labeled DNA oligonucleotides gave characteristic EPR spectra and caused the disappearance of bound protein signals in N-15 HSQC spectra consistent with the paramagnetic relaxation enhancement (PRE) effect. (C) 2015 The Authors. Published by Elsevier Ltd.

引用

页码：813 / 819

页数：7

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