Single-molecule chemistry. Part I: monitoring oxidation of G in oligonucleotides using CY3 fluorescence
Open Access
- 19 May 2020
- journal article
- research article
- Published by IOP Publishing in Methods and Applications in Fluorescence
- Vol. 8 (3), 035010
- https://doi.org/10.1088/2050-6120/ab947d
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 photo- induced 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.Keywords
This publication has 108 references indexed in Scilit:
- A rule of seven in Watson-Crick base-pairing of mismatched sequencesNature Structural & Molecular Biology, 2012
- A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomersNucleic Acids Research, 2012
- The Formamidopyrimidines: Purine Lesions Formed in Competition With 8-Oxopurines From Oxidative StressAccounts of Chemical Research, 2011
- Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible TethersBiophysical Journal, 2011
- Cy3-DNA Stacking Interactions Strongly Depend on the Identity of the Terminal BasepairBiophysical Journal, 2011
- Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence ImagingBiophysical Journal, 2009
- Real-Time DNA Sequencing from Single Polymerase MoleculesScience, 2009
- Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acidsProceedings of the National Academy of Sciences of the United States of America, 2008
- An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence ExperimentsBiophysical Journal, 2008
- Photoinduced Electron Transfer in Ethidium-Modified DNA Duplexes: Dependence on Distance and Base StackingJournal of the American Chemical Society, 1997