Single-molecule chemistry. Part I: monitoring oxidation of G in oligonucleotides using CY3 fluorescence. (30th June 2020)

• Record Type: Journal Article
• Authors: Sobek, Jens; Schmidt, Marco; Grossmann, Jonas; Rehrauer, Hubert; Schmidt, Lucas; Schlapbach, Ralph
• Is Part Of: Methods and applications in fluorescence. Volume 8:Number 3(2020:Sep.)
• Publication Date: 2020-06-30
• Subjects: single-molecule hybridisation -- fluorescence quenching -- short oligonucleotides -- CY3 -- guanine oxidation -- 8-oxoG -- single-molecule chemistry; Fluorescence spectroscopy -- Periodicals; Fluorescence -- Periodicals; Spectrometry, Fluorescence -- Periodicals; Fluorescence -- Periodicals; Fluorescence; Fluorescence spectroscopy; Periodicals; 535.352
• DOI: 10.1088/2050-6120/ab947d
• Languages: English
• ISSNs: 2050-6120

Abstract

Abstract: 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.