University of Iceland, Science Institute, Dunhaga 3, 107 Reykjavik, Iceland.
J Am Chem Soc. 2009 Dec 23;131(50):18054-6. doi: 10.1021/ja905623k.
The spin-labeled nucleoside (T)C, 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 ((T)C.G) and the mismatches ((T)C.A, (T)C.C, and (T)C.T) were significantly different, showing that the probe can identify its base-pairing partner in DNA. At lower pH, the mobility of (T)C.A, (T)C.C, and (T)C.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(T)CA) and 5'-d(T(T)CA). This study shows that minor structural variations in nucleic acids can be detected with carefully chosen spin labels in conjunction with EPR spectroscopy.
首次通过电子顺磁共振（EPR）光谱法，使用与 C 的环外氨基共轭的 2,2,6,6-四甲基哌啶-1-氧自由基（TEMPO）标记核苷（T）C 来检测双链 DNA 中的单碱基错配。此外，完全碱基配对的双链（（T）C.G）和错配（（T）C.A、（T）C.C 和（T）C.T）的 EPR 谱有明显差异，表明探针可以识别其在 DNA 中的碱基配对伙伴。在较低的 pH 值下，（T）C.A、（T）C.C 和（T）C.T 的迁移率增加，这与错配碱基对的质子化增加一致。尽管测试的每个侧翼序列的双链都给出了可区分的 EPR 信号，但对于包含侧翼 A.T 碱基对的序列，特别是 5'-d（G（T）CA）和 5'-d（T（T）CA），碱基对之间的区分效果最好。本研究表明，通过精心选择的自旋标记物结合 EPR 光谱法，可以检测核酸中的微小结构变化。
