Type of presentation: Poster

LS-3-P-3193 UV-activated conversion of Hoechst and DAPI to their green-emitting protonated forms and their application in super-resolution microscopy

Żurek-Biesiada D.1, Szczurek A.2, Waligórski P.3, Prakash K.2, Cremer C.2,4, Dobrucki J.1
1Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 2Institute of Molecular Biology (IMB), Mainz, Germany, 3The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland, 4Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, Germany
dominika.zurek@uj.edu.pl

KEY-WORDS: DNA-binding dyes, Hoechst, DAPI, photoconversion, protonation, super-resolution microscopy

BACKGROUND. In our recent paper we have reported that, upon excitation with UV, DNA-binding dyes Hoechst 33258 and DAPI undergo photoconversion [1]. Although a typical loss of fluorescence (photobleaching) is observed, apparently a fraction of the population of the blue-emitting form of the dye do not lose the ability to fluoresce but is are converted into a blue-excited, green-emitting form. Mass spectrometry data suggest that the observed spectral changes are associated with UV-induced protonation of the Hoechst molecule [2].

Goal. The purpose of this work is to shed more light on the photophysics of the process of UV-activated conversion of the DNA-binding dyes and to apply those dyes in super-resolution microscopy.

Methods. Spectrofluorimetry, mass spectrometry, spectrally-resolved confocal microscopy, SPDM

RESULTS and CONCLUSION. We demonstrate that the UV-generated form of Hoechst 33258 presents different spectral characteristics, with its absorption and emission spectrum shifted towards longer wavelengths. The photoproduct is stable in time, UV-dose dependent and the process of photoconversion does not require the presence of the DNA. Moreover, Hoechst 33258 exhibits the same spectral properties as the forms of Hoechst that can be obtained by subjecting the dye to a highly acidic environment (pH 0.5-3.0). By using mass spectrometry and spectrofluorimetry we demonstrate that exposing Hoechst to UV leads to generation of three protonated forms of the dye. The spectral properties and affinity to nucleic acids of these protonated forms differ from the original blue-emitting form. Our recent findings suggest that Hoechst may be used as a DNA probe in super-resolution microscopy (Szczurek et al., submitted). The key to a successful exploitation of the phenomenon of photoconversion is understanding of the photophysics of this process.

Reference
[1] Zurek-Biesiada D, Kędracka-Krok S, Dobrucki JW. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry A 83(5):441-51 (2013).
[2] Żurek-Biesiada D, Waligórski P, Dobrucki J. Mass spectrometry and fluorimetry analysis of blue-excited green-emitting protonated forms of Hoechst 33258 generated by UV illumination (in preparation).
[3] Szczurek A, Prakash K; Lee HK, Żurek-Biesiada D; Best G, Hagmann M, Dobrucki J, Cremer C, Birk U. Single molecule localisation microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes (manuscript submitted).