Rhodamine-Hoechst positional isomers for highly efficient staining of heterochromatin.

Estimation of absorbance and fluorescence increase upon

SDS or hpDNA addition Fluorescence increase of the probes upon SDS or hpDNA addition was measured by preparing 2 μM probe solution in PBS buffer (Lonza, Cat. No. BE17-516F) with or without 0.1% SDS (Acros Organics). The samples prepared in 3 ml glass bottles with caps were incubated at room temperature for 2 h before measurements. The fluorescence increase of Hoechst-based probes binding to hpDNA was estimated using the following procedure: the probe from 1 mM DMSO stock solution was diluted to the final concentration of 2 μM in PBS buffer containing 30 μM of hpDNA (5′-CGCGAATTCGCGTTTTCGCGAATTCGCG-3′). Absorption and fluorescence were measured on a multiwell plate reader Spark® 20M (Tecan) in glass bottom 96-well plates (MatTek, Cat. No. PBK96G-1.5-5-F) at room temperature (25 °C). Absorption of solutions was recorded from 320 nm to 850 nm with a wavelength step size of 1 nm. The background absorption of the glass bottom plate was measured in wells containing only buffer and subtracted from the spectra of the samples. The fluorescence emission of the free dyes or final probes was recorded from 520 nm to 850 nm (for TMR, 495 nm exc., bandwidth 15 nm), 560 nm to 850 nm (for 580CP, 530 nm exc., bandwidth 15 nm), 600 nm to 850 nm (for 610CP, 570 nm exc., bandwidth 15 nm), and 620 nm to 850 nm (for SiR/GeR, 595 nm exc., bandwidth 15 nm) with a 5 nm emission bandwidth and 2 nm step size. All samples were prepared in technical triplicates, which was repeated three times as three independent experiments performed on different days.

Determination of quantum yields and lifetimes

The fluorescence quantum yields (absolute values) were obtained with a Quantaurus-QY absolute PL quantum yield spectrometer (model C11347-12, Hamamatsu) according to the manufacturer's instructions. Fluorescence lifetimes were measured with a Quantaurus-Tau fluorescence lifetime spectrometer (model C11367-32, Hamamatsu) according to the manufacturer's instructions. All measurements were performed in air-saturated PBS buffer containing 2 μM probe and 30 μM hpDNA after incubation for 2 h at room temperature.

Determination of K d

K d measurements were performed by titrating Hoechst or its derivatives in PBS (Lonza) with increasing concentrations of the 28 bp hpDNA in a 96-well plate and measuring the increase in fluorescence on a plate reader after 1 h incubation at room temperature. Hoechst 33342, TMR, 580CP, 610CP, GeR and SiR dyes were excited at 360 nm, 540 nm, 570 nm, 570 nm, 640 nm and 640 nm while recording emission at 480 nm, 580 nm, 610 nm, 640 nm, 670 nm and 670 nm respectively. The excitation and emission bandwidths were 10 nm for SiR/GeR and 15 nm for all other dyes. The K d values were determined by plotting the emission signal vs. hpDNA concentration and fitting the curve in GraphPad Prism 6 to the “Two site-binding + offset” function: 1 where A 1 and A 2 – fluorescence change after probe binding to the 1st and 2nd site respectively, X – target hpDNA concentration, K d1 and K d2 – dissociation constant of the probe from the 1st and 2nd site respectively, and offset – background fluorescence signal or “Single site binding” function: 2 where F min – fluorescence of the probe without the target, F max – fluorescence of the probe at the saturating concentration, p – probe concentration, X – target hpDNA concentration, and K d – dissociation constant of the probe. All measurements performed 3 times on different days, each time technical triplicates were measured. STED microscope with a 775 nm depletion laser All confocal and STED images were acquired on an Abberior STED 775 QUAD scanning microscope (Abberior Instruments GmbH, Germany) equipped with 561 nm and 640 nm 40 MHz pulsed excitation lasers, a pulsed 775 nm 40 MHz STED laser, and an UPlanSApo 100×/1.40 Oil objective. The following detection windows were used: TMR/Cy3 channel 615/20 nm and Cy5 channel 685/70 nm. The voxel size was 15–30 nm in the xy plane and 150 nm in the z -axis for STED images acquired using this setup. The pixel size was 50–150 nm in the xy plane for confocal images acquired using this setup. Laser powers were optimized for each sample. Molecular docking The DNA-Hoechst 33258 complex structure was downloaded from the PDB database (PDB ID: 8BNA ). 11 Only DNA molecules were used for the docking experiments. Ligands were drawn using ChemDraw Professional 15.1 and prepared for docking with AutoDock Tools version 1.5.6 ( ref. 12 ). The docking simulation was performed using Vina Autodock version 1.1.2 ( ref. 13 ). Twenty binding modes were generated starting from random configurations of ligands that had fully flexible torsional degrees of freedom.

Image analysis

The methods used for the image analysis are described in the ESI. †

Supplementary Material Supplementary information Click here for additional data file. Supplementary movie Click here for additional data file. Supplementary movie Click here for additional data file.