Functional Genomics Center Zurich, Eidgenössische Technische Hochschule (ETH) Zurich and University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Methods Appl Fluoresc. 2020 Jul 7;8(3):035010. doi: 10.1088/2050-6120/ab947d.
Single-molecule hybridisation of CY3 dye labelled short oligonucleotides to surface immobilised probes was investigated in zero-mode waveguide nanostructures using a modified DNA sequencer. At longer measuring times, we observed changes of the initial hybridisation fluorescence pulse pattern which we attribute to products created by chemical reactions at the nucleobases. The origin is a charge separated state created by a photoinduced electron transfer from nucleobases to the dye followed by secondary reactions with oxygen and water, respectively. The positive charge can migrate through the hybrid resulting in base modifications at distant sites. Static fluorescence spectra were recorded in order to determine the properties of CY3 stacking to different base pairs, and compared to pulse intensities. A characteristic pulse pattern change was assigned to the oxidation of G to 8-oG besides the formation of a number of secondary products that are not yet identified. Further, we present a method to visualise the degree of chemical reactions to gain an overview of ongoing processes. Our study demonstrates that CY3 is able to oxidise nucleobases in ds DNA, and also in ss overhangs. An important finding is the correlation between nucleobase oxidation potential and fluorescence quenching which explains the intensity changes observed in single molecule measurements. The analysis of fluorescence traces provides the opportunity to track complete and coherent reaction sequences enabling to follow the fate of a single molecule over a long period of time, and to observe chemical reactions in real-time. This opens up the opportunity to analyse reaction pathways, to detect new products and short-lived intermediates, and to investigate rare events due to the large number of single molecules observed in parallel.
在零模式波导纳米结构中，使用改良的 DNA 测序仪研究了 CY3 染料标记的短寡核苷酸与表面固定探针的单分子杂交。在较长的测量时间内，我们观察到初始杂交荧光脉冲模式的变化，我们将其归因于核碱基处化学反应产生的产物。其起源是光诱导电子从核碱基转移到染料后产生的电荷分离态，随后分别与氧和水发生二次反应。正电荷可以通过杂交迁移，导致远程位置的碱基修饰。记录静态荧光光谱以确定 CY3 与不同碱基对堆叠的性质，并与脉冲强度进行比较。特征脉冲模式的变化被分配给 G 到 8-oG 的氧化，除了形成一些尚未确定的次级产物之外。此外，我们提出了一种可视化化学反应程度的方法，以获得正在进行的过程的概述。我们的研究表明，CY3 能够氧化 dsDNA 中的核碱基，也能够氧化 ss 突出端。一个重要的发现是核碱基氧化电位与荧光猝灭之间的相关性，这解释了在单分子测量中观察到的强度变化。荧光轨迹的分析提供了跟踪完整和连贯反应序列的机会，从而能够在很长一段时间内跟踪单个分子的命运，并实时观察化学反应。这为分析反应途径、检测新产物和短暂中间体以及研究由于同时观察到大量单个分子而导致的罕见事件提供了机会。
