Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.

Autor:	Bachman JL; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Wight CD; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Bardo AM; Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States., Johnson AM; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Pavlich CI; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Boley AJ; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Wagner HR; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Swaminathan J; Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States., Iverson BL; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States., Marcotte EM; Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States., Anslyn EV; Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.
Jazyk:	angličtina
Zdroj:	Bioconjugate chemistry [Bioconjug Chem] 2022 Jun 15; Vol. 33 (6), pp. 1156-1165. Date of Electronic Publication: 2022 May 27.
DOI:	10.1021/acs.bioconjchem.2c00103
Abstrakt:	A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG) 10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.
Databáze:	MEDLINE
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