Functional Genomics Center Zurich, Eidgenössische Technische Hochschule (ETH) Zurich and University of Zurich, 8057 Zurich, Switzerland.
Molecules. 2020 Nov 17;25(22):5369. doi: 10.3390/molecules25225369.
To understand the complex fluorescence properties of astraphloxin (CY3)-labelled oligonucleotides, it is necessary to take into account the redox properties of the nucleobases. In oligonucleotide hybrids, we observed a dependence of the fluorescence intensity on the oxidation potential of the neighbouring base pair. For the series I < A < G < 8-oxoG, the extent of fluorescence quenching follows the trend of decreasing oxidation potentials. In a series of 7 nt hybrids, stacking interactions of CY3 with perfect match and mismatch base pairs were found to stabilise the hybrid by 7-8 kJ/mol. The fluorescence measurements can be explained by complex formation resulting in fluorescence quenching that prevails over the steric effect of a reduced excited state trans-cis isomerisation, which was expected to increase the fluorescence efficiency of the dye when stacking to a base pair. This can be explained by the fact that, in a double strand, base pairing and stacking cause a dramatic change in the oxidation potential of the nucleobases. In single-molecule fluorescence measurements, the oxidation of G to 8-oxoG was observed as a result of photoinduced electron transfer and subsequent chemical reactions. Our results demonstrate that covalently linked CY3 is a potent oxidant towards dsDNA. Sulfonated derivatives should be used instead.
为了理解天青石素（CY3）标记寡核苷酸的复杂荧光性质，有必要考虑核碱基的氧化还原性质。在寡核苷酸杂交体中，我们观察到荧光强度与相邻碱基对的氧化电位有关。对于 I<A<G<8-oxoG 系列，荧光猝灭的程度遵循氧化电位降低的趋势。在一系列 7nt 杂交体中，发现 CY3 与完全匹配和错配碱基对的堆积相互作用通过 7-8kJ/mol 稳定杂交体。荧光测量可以通过复合物形成来解释，导致荧光猝灭超过减少的激发态 trans-cis 异构化的空间位阻效应，这预计会增加染料的荧光效率当堆积到碱基对时。这可以解释为在双链中，碱基配对和堆积导致核碱基的氧化电位发生剧烈变化。在单分子荧光测量中，观察到 G 氧化为 8-oxoG 是由于光诱导电子转移和随后的化学反应所致。我们的结果表明，共价连接的 CY3 是 dsDNA 的有效氧化剂。应使用磺化衍生物代替。
