Identification of Single-Base Mismatches in Duplex DNA by EPR Spectroscopy

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• 作者：Pavol Cekan ; Snorri Th. Sigurdsson
• 刊名：Journal of the American Chemical Society
• 出版年：2009
• 出版时间：December 23, 2009
• 年：2009
• 卷：131
• 期：50
• 页码：18054-18056
• 全文大小：235K
• 年卷期：v.131,no.50(December 23, 2009)
• ISSN：1520-5126

文摘

The spin-labeled nucleoside ^TC, containing 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) conjugated to the exocyclic amino group of C, was used to detect single-base mismatches in duplex DNA for the first time by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, the EPR spectra of the fully base-paired duplex (^TC·G) and the mismatches (^TC·A, ^TC·C, and ^TC·T) were significantly different, showing that the probe can identify its base-pairing partner in DNA. At lower pH, the mobility of ^TC·A, ^TC·C, and ^TC·T became higher, consistent with increased protonation of the mismatched pairs. Although the duplexes for each of the three flanking sequences tested gave distinguishable EPR signals, the best discrimination between base pairs was achieved for sequences containing a flanking A·T base pair, in particular 5′-d(G^TCA) and 5′-d(T^TCA). This study shows that minor structural variations in nucleic acids can be detected with carefully chosen spin labels in conjunction with EPR spectroscopy.