Cell permeable fluorescent DNA dyes that allows visualization of chromatin in many cell types without transfection are highly required by cell biologist and other scientists. Such fluorescent probes gives the invaluable opportunity to observe chromatin in the cells of many different organisms. Hoechst and DAPI dyes are amongst most commonly used DNA stains, however they are known to cause DNA damage. A recently developed DNA probe SiR-Hoechst (aka SiR-DNA) is reported to cause low toxicity, has been commercialised and widely used by many laboratories. However, staining of a number of cell lines revealed its weak points: efflux from cells by multidrug resistance pumps and relatively low brightness compared to the original Hoechst 33342.<br/>Hoechst conjugates to fluorescent dyes became popular DNA stains for live-cell imaging, but the relationship between their structure and performance remains elusive. This study of carboxyrhodamine–Hoechst 33258 conjugates reveals that a minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6’-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5’-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Employing the highly efficient probes for two-color STED allowed visualization of DNA and tubulin structures in intact nucleated erythrocytes – a system where imaging is greatly hampered by high haemoglobin absorbance.

Rhodamine–Hoechst positional isomers for highly efficient staining of heterochromatin

2019 Fall National Meeting

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