Rhodamine–Hoechst positional isomers for highly efficient staining of heterochromatin

Nuclei of living cells fluoresce brighter after staining with rhodamine–Hoechst 5-isomers.


Supplementary inventory
. Photophysical properties of rhodamine fluorescent dyes used in the study. .... 21 Table S2.      Coordinates taken from PDB ID: 8BNA 1 . Docking result shows possible conformations of the probes: all conformers of isomer-5 are positioned in the minor groove, but some of the isomer-6 conformers are predicted to be positioned in the major groove.
Video S2. Rotating maximum intesity projection of the intact Xenopus laevis erythocyte. The sample of whole blood was stained with 1µM SiR-CTX (red, tubulin) and 1 µM 5-580CP-Hoechst (green, nucleus) at RT for 1 h in the RBC buffer. No washing step was applied. Aquired z-stack was deconvolved using SVI Huygens.  Note: a For the spectroscopy studies hairpin-forming oligonucleotide 5'-CGCGAATTCGCGTTTTCGCGAATTCGCG were used; probe concentration -2µM, oligonucleotide concentration -30 µM; probes were incubated for 2h at room temperature before the measurements. Data presented as mean value with standart deviation, N = 3. b Extinction coeficients of probes were equated to the maximal extinction coficients of fluorescent dyes

Estimation of the probe aggregation
The estimation of aggregation extent was performed using a centrifugation-based assay. The 2 µM solutions of probes were prepared by adding 1mM DMSO stock solution to PBS (Lonza,Cat. no. BE17-516F). After 1 h incubation at RT, the solutions were centrifuged for 1h at 20 o C in a centrifuge set at 16000g. The pellet and solution fractions were separated, the pellet was suspended in PBS + 0.1% SDS and the supernatant was supplemented with SDS to 0.1% concentration. After 1h incubation at RT, the pellet was fully dissolved, and fluorescence was measured for both supernatant and pellet fractions with a multiwell plate reader Spark® 20M (Tecan) in a glass bottom 96-well plate (MatTek, Cat. no. PBK96G-1.5-5-F) at room temperature. The previously described settings were used for excitation and emission. Aggregation percentage was calculated using the following formula S1:

Preparation of erythrocytes
Sodium citrate treated whole blood from frog or chicken was diluted 1:500 with RBC buffer (

Processing and visualization of acquired images
All acquired or reconstructed images were processed and visualized using Fiji 5 . Line profiles were measured using the "straight line" tool with the line width set to 3 pixels.
For the signal measurements, image files were converted to TIF file using Fiji and analyzed with CellProfiler 3.1.5 (ref. 6 ), where the pipeline identified the nuclear region and measured the mean signal in this region. Background signal was measured in the region which is 3 pixels (450 nm) away from the nulear border and 7 pixels (1050 nm) wide. The background substracted signal was processed with GraphPad Prism 6.
The estimation of the diameter of heterochromatin exclusion zones near nuclear pore complexes was performed with custom written Matlab (Mathworks, Natick, US) routines:

Correction of drift during image acquisition
Frame to frame pixel shifts were estimated by computing all possible 2D cross-correlations between frames of the 3D data stacks that were not more than 450nm apart (well within the FWHM of the PSF along z). The pixel shift was taken as the position of the maximum amplitude of a 2D cross-correlation relative to the origin of the coordinate system.
Then a smooth drift curve was estimated, independently for x and y directions, that realizes the estimated pixel shifts as closely as possible. The minimization metric was a weighted least square distance between drift and estimated pixel shifts. An additional regularization term proportional to the total variation of the drift was introduced that additionally smoothed the drift curve. The drift in the data was then corrected by shifting data frames by whole pixels in the opposite direction of the estimated drift.

Deconvolution of 3D image z-stacks
Drift corrected image stacks were deconvolved with a measured point spread function (PSF) using the Richardson-Lucy algorithm 7 for 10-20 iterations on each measured 3D data set. The Then randomly 15 patches were selected repeatedly, averaged to deliver a more symmetric hole with a flat surrounding and a symmetric 2D Gaussian peak was fitted to it using least squares minimization. The estimated FWHM was recorded and the procedure was repeated multiple times. If n patches were available for a particular condition 20n/log(n) times 15 patches were drawn randomly and the average image of these patches was fitted. Biotage Isolera flash purification system using the type of cartridge and solvent gradient indicated. (50 mL), extracted with EtOAc (3×50 mL), the combined organic layers were washed with water, brine and dried over Na2SO4. After filtration, the solvent was evaporated to give the crude solid diamide, which was used directly in the next step.

5-SiR-COOH
To a solution of bis-oxazoline SI-1 (606 mg, 1.73 mmol, 2 equiv) in THF (15 mL), cooled to -78°C, t-BuLi (2.04 mL of 1.7 M in pentane, 3.46 mmol, 4 equiv) was added, and the mixture was stirred at -78°C for 1 h. A solution of ketone SI-2 9 (280 mg, 0.863 mmol) in THF (20 mL) was added dropwise, and the mixture was allowed to warm up to r.t. and stirred for 3 h. Acetic acid (2 mL) was added in to the brownish reaction mixture which immediately turned dark blue.
Mixture was evaporated on a rotary evaporator to a viscous residue, which was then dissolved in 6 N HCl (40 mL), and the resulting orange-brown solution was stirred at 80°C overnight.
The yellow mixture was cooled and pH was adjusted to 1-2 by careful addition of NaHCO3.
The mixture was extracted with CH2Cl2 (5×50 mL), the poorly soluble dye was dissolved by addition of methanol to the combined organic layers, which were then dried over Na2SO4, filtered and evaporated. The product was isolated by flash column chromatography (Büchi Reveleris HP silica 40 g, gradient 20% to 100% hexane -EtOAc with constant 1% v/v AcOH adittive ), the fractions containing the product were evaporated, the residue was redissolved

5-610CP-COOH
To a solution of bis-oxazoline SI-1 (233 mg, 0.65 mmol, 2 equiv) in THF (5 mL), cooled to -78°C, t-BuLi (0.8 mL of 1.7 M in pentane, 1.36 mmol, 4 equiv) was added, and the mixture was stirred at -78°C for 1 h, gradually turning light orange. A solution of ketone SI-3 (ref. 10 ) (101 mg, 0.33 mmol) in THF (5 mL) was added dropwise, and the mixture was allowed to warm up to r.t. and stirred for 2 h. The resulting brownish solution was cooled in ice-water bath, and acetic acid (1 mL) was added. The dark blue reaction mixture was evaporated on a rotary evaporator to a viscous residue, which was dissolved in 6 N HCl (20 mL), and the resulting dark orange solution was stirred at 80°C overnight. The yellow mixture was cooled and pH was adjusted to 1-2 by careful addition of NaHCO3. The mixture was extracted with CH2Cl2 (5×50 mL), the poorly soluble dye was dissolved by addition of methanol to the combined organic layers, which were then dried over Na2SO4, filtered and evaporated. The product was isolated by flash column chromatography (Büchi Reveleris HP silica 24 g, gradient 40% to 100% hexane -EtOAc with constant 1% v/v AcOH adittive), the fractions containing the product were evaporated, the residue was redissolved in 1,4-dioxanewater
The cooling bath was removed, the mixture was allowed to warm up to room temperature and stirred for further 30 min. The reaction mixture was quenched with water (10 mL), adjusted to pH ~ 5 with acetic acid, extracted with ethyl acetate (3×30 mL), the combined organic layers were washed with brine and dried over Na2SO4. The product was isolated by flash column chromatography (Büchi Reveleris HP silica; gradient 0% to 20% ethyl acetatehexane) as white solid, yield 292 mg (41%).
The reaction mixture was then evaporated to dryness, the residue was re-evaporated three times with toluene to remove excess trifluoroacetic acid. The residue was lyophilized from aqueous dioxane. Product obtained as trifluoroacetic acid salt, yield 120 mg (98%), purpleblue solid.

5-GeR-COOH
To a solution of bis-orthoester SI-10 12 (111 mg, 0.27 mmol, 2 equiv) in THF (10 mL), cooled to -78°C, t-BuLi (320 µL of 1.7 M in pentane, 0.54 mmol, 4 equiv) was added, and the mixture was stirred at -78°C for 1 h. A solution of ketone SI-11 13 (50 mg, 0.135 mmol) in THF (5 mL) was added dropwise, and the mixture was allowed to warm up to r.t. and stirred for 2 h. The resulting solution was cooled in ice-water bath, and acetic acid (1 mL) was added and stirred for 10 minutes. The dark blue reaction mixture was evaporated on a rotary evaporator to a viscous residue, which was dissolved in 6 N HCl (20 mL), and the resulting orange-brown solution was stirred at 80°C overnight. The yellow mixture was cooled and pH was adjusted to 1-2 by careful addition of NaHCO3. The mixture was extracted with CH2Cl2 (5×50 mL), the poorly soluble dye was dissolved by addition of methanol to the combined organic layers, which were then dried over Na2SO4, filtered and evaporated. The product was isolated by flash column chromatography (Büchi Reveleris HP silica 24 g, gradient 20% to 80% hexane -EtOAc with constant 1% v/v AcOH adittive), the fractions containing the product were evaporated, the residue was redissolved in acetonitrilewater (1:1), microfiltered through a 0.45 μm PTFE membrane filter and lyophilized. Yield 35 mg (68%), blue solid.