Super‐Resolution Spatial Proximity Detection with Proximity‐PAINT

Abstract Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single‐molecule sensitivity. Here, we introduce Proximity‐PAINT (pPAINT), a variation of the super‐resolution microscopy technique DNA‐PAINT. pPAINT uses a split‐docking‐site configuration to detect spatial proximity with high sensitivity, low false‐positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha‐ and beta‐tubulin in microtubules using super‐resolution detection.


Table of Contents
Quantitative pPAINT measurements Stem = 9nt (corresponding to Fig. 1e and Figure S6) Supplementary Table 6 Quantitative pPAINT measurements Stem = 10nt (corresponding to Figure S6) Supplementary  Both photostabilization systems allowed us to maximize the number of photons per event and thus achieve optimal spatial resolution.
DNA origami self-assembly. All DNA origami structures were designed with the Picasso [1] design tool (see Figure S1). Self-assembly of DNA origami was accomplished in a one-pot reaction mix with 50 μl total volume, consisting of 10 nM scaffold strand (sequence see Data S1), 100 nM folding staples (Data S2-S4), 10 nM biotinylated staples (Table S10), and 1 μM of docking site strands (List of DNA-PAINT handles see Table S7 & S8) in folding buffer (1× TE buffer with 12.5 mM MgCl2). The reaction mix was then subjected to a thermal annealing ramp using a thermocycler. The reaction mix was first incubated at 80 °C for 5min and then immediately cooled down to 60 ºC. Subsequently, the sample was cooled from 60 to 4 °C in steps of 1 °C per 3.21 min and then held at 4 °C.
DNA origami sample preparation. For sample preparation of Figure 1b-e and Figure S3, a µ-Slide VI 0.5 from ibidi was used as sample chamber. First, 100 μl of biotin labeled bovine albumin (1 mg/ml, dissolved in buffer A) was flushed into the chamber and incubated for 5 min. The chamber was then washed with 500 μl of buffer A. A volume of 100 μl of streptavidin (0.5 mg/ml, dissolved in buffer A) was then flushed through the chamber and allowed to bind for 5 min. After washing with 500 μl of buffer A and subsequently with 500 μl of buffer B, 100 μl of biotin labeled DNA structures (~200 pM) in buffer B were flushed into the chamber and incubated for 8 min. The chamber was washed with 500 μl of buffer B. Finally, 100 μl of the imager solution in the corresponding imaging buffer (see Table S12) was flushed into the chamber. For sample preparation of Figure 1f and Figure S4-S6, a piece of coverslip and a glass slide were sandwiched together by two strips of double-sided tape to form a flow chamber with inner volume of ~20 μl. First, 20 μl of biotin labeled bovine albumin (1 mg/ml, dissolved in buffer A) was flushed into the chamber and incubated for 2 min. The chamber was then washed with 40 μl of buffer A. A volume of 20 μl of streptavidin (0.5 mg/ml, dissolved in buffer A) was then flushed through the chamber and allowed to bind for 2 min. After washing with 20 μl of buffer A and subsequently with 20 μl of buffer B, 20 μl of biotin labeled DNA structures (~200 pM) in buffer B were flushed into the chamber and incubated for 2 min. The chamber was washed with 40 μl of buffer B. Finally, 20 μl of the imager solution in the corresponding imaging buffer (see Table S12) was flushed into the chamber, which was subsequently sealed with two component silica before imaging.
Antibody conjugation. Antibodies were conjugated to DNA-PAINT docking sites via maleimide-PEG2-succinimidyl ester chemistry as previously reported [1] . Cell culture. U-2 OS-CRISPR-Nup96-mEGFP cells were passaged every other day and used between passage number 5 and 20. The cells were maintained in McCoy's 5A medium supplemented with 10 % Fetal Bovine Serum. Passaging was performed using 1× PBS and Trypsin-EDTA 0.05 %. 24 h before immunostaining, cells were seeded on Eppendorf 8-well glass coverslips at 30,000 cells/well.

Cell fixation.
For fixation, the samples were fixed and permeabilized with 3 % formaldehyde, 0.1 % glutaraldehyde and 0.25 % Triton X-100 for 12 min. Next, samples were rinsed twice (5 min) with 1× PBS and then quenched with 0.1 % NaBH4 for 7 min. After rinsing four times with 1× PBS for 30 s, 60 s, and twice for 5 min, samples were blocked and permeabilized with 3 % BSA and 0.25 % Triton X-100 for 2 h. Then, samples were incubated with 10 μg/ml of primary antibodies (1:200 dilution) in a solution with 3 % BSA and 0.1 % Triton X-100 at 4 °C overnight. Cells were washed three times (5 min each) with 1× PBS. Next, they were incubated with 10 μg/ml of labeled secondary antibodies (1:100 dilution) in a solution with 3 % BSA and 0.1 % Triton X-100 at room temperature for 2 hours. For fiducial based drift correction, the samples were incubated with gold nanoparticles with a 1:1 dilution in 1× PBS for 5 min. Finally, samples were rinsed three times with 1× PBS before adding imager solution.
Super-resolution microscope. Fluorescence imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100×, NA 1.49, Oil). A 561 nm (MPB Communications Inc., 2 W, DPSS-system) laser was used for excitation. The laser beam was passed through cleanup filters (Chroma Technology, ZET561/10) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m and ET575lp) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2×2 binning). Figure 1b-e. First round of imaging was carried out using an imager strand concentration of 7.5 nM (pPS) and 2.5 nM (P3) in imaging buffer (see Table S12). 20,000 frames were acquired at 100 ms exposure time. The readout bandwidth was set to 200 MHz. Laser power (@561 nm) was set to 20 mW (measured before the back focal plane (BFP) of the objective), corresponding to 113 W/cm 2 at the sample plane. After imaging the sample was subsequently washed five times with 100 µl each with 1× PBS (on the microscope). Second round of imaging was carried out using an imager strand concentration of 2.5 nM (P3) and 2.5 nM (P6) in imaging buffer (see Table S12). 5,000 frames were acquired at 100 ms exposure time. The readout bandwidth was set to 200 MHz. Laser power (@561 nm) was set to 100 mW (measured before the back focal plane (BFP) of the objective), corresponding to 564 W/cm 2 at the sample plane. Figure 1f. Images were acquired with an imager strand concentration of 5 nM (pPS) in imaging buffer (see Table S12). 20,000 frames were acquired at 300 ms exposure time. The readout bandwidth was set to 200 MHz. Laser power (@561 nm) was set to 100 mW (measured at the back focal plane (BFP) of the objective). corresponding to 564 W/cm 2 at the sample plane.  Table S12). 20,000 frames were acquired at 50 ms exposure time and a readout bandwidth of 200 MHz. Laser power (@560 nm) was set to 90 mW (measured before the back focal plane (BFP) of the objective), corresponding to 508 W/cm 2 at the sample plane. Image analysis. Raw fluorescence data was subjected to spot-finding and subsequent super-resolution reconstruction using the 'Picasso' software package [1] . x, y and z drift correction was performed with a redundant cross-correlation and DNA origami or gold particles as fiducials. Quantitative analysis. pPAINT data (Figure 1b-e, Figure S3) was selected based on the method described in Figure S2. The data was linked (gap size = 4 frames), and afterwards filtered. As filter criteria the 'mean frame' with 10% around the maximum, the 'std frame' >10% and the number of localizations with 15 < 'n events' < 75 were applied.    Table S5). (b) Distance measurements for d = 0 nm (red), d = 5 nm (magenta), d = 10 nm (blue) and d = 20 nm (green) for different leash lengths (poly-T, x-axis) and a stem of 10 nt (see Table S6).     [2] .