DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates.

Probe synthesis and purification

All oligonucleotides (Supplementary Table 1 , Supplementary Fig. 1 ) were custom synthesized by Integrated DNA Technologies, except for BHQ1 modified oligos, which were custom synthesized by Biosearch Technologies. One hundred micrograms cyclo [Arg-Gly-Asp-D-Phe-Lys(PEG-PEG)] (PCI-3696-PI, Peptides International) was sonicated with 50 μg NHS-azide (88902, Thermo Fisher Scientific) in 10 μL dimethyl sulfoxide (MX1457-7, Millipore-Sigma) for 1 h. The azide-modified cyclo [Arg-Gly-Asp-D-Phe-Lys(PEG-PEG)] peptide was purified via reverse-phase high performance liquid chromatograph (HPLC) with a Grace Alltech C18 column (0.75 mL min −1 flow rate; solvent A: nanopure water + 0.05% trifluoracetic acid (TFA), solvent B: acetonitrile (BDH83639.400, VWR) + 0.05% TFA; starting condition 90% A + 10% B, 1% per min gradient B) (Supplementary Fig. 2a ). Following HPLC purification, products were dried in an Eppendorf Vacufuge plus. Subsequently, the azide-modified peptide was ligated to DNA oligos containing a 5′ hexynyl modification using copper-catalyzed azide-alkyne cycloaddition. Briefly, 5 μL of 1 mM oligonucleotide was reacted for 1 h with ~30 nmol azido peptide in the presence of 10 mM sodium ascorbate, 1 mM copper sulfate, and 0.8 mM THPTA (1010, Click Chemistry Tools). The reaction was purged under N 2 . The product was purified using reverse-phase HPLC with an Agilent Advanced oligo column (0.5 mL min −1 flow rate; solvent A: 0.1 M TEAA, solvent B: acetonitrile; starting condition: 90% A + 10% B, 1% per min gradient B). Oligos were conjugated to Cy3B NHS ester (PA63100, GE Healthcare) in a 10 μL reaction; 50 μg excess Cy3B NHS was reacted with 2–5 nmol amine-modified DNA in 1× phosphate buffered saline (PBS) and 0.1 M sodium bicarbonate overnight. The product was purified using a P2 size-exclusion gel to remove excess dye prior to HPLC purification. Reverse-phase HPLC was performed with an Agilent Advanced oligo column as described above (Supplementary Fig. 2b–d ). Final products were resuspended in nanopure water. If significant excess dye remained for MT-FLIM strands, the DNA was repurified with an amicon filter (Amicon Ultra-0.5 mL, Centrifugal Filters, Ultracel-3K) or with HPLC. Starting material and final masses were confirmed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) (Supplementary Fig. 3 , Supplementary Table 2 ).

Mass spectrometry

Oligonucleotides in nanopure water (18.2 M Ω, Barnstead Nanopure) were plated in a 1:1 vol/vol ratio with saturated 3-hydroxypicolinic acid (56197, Millipore-Sigma) in 50% acetonitrile, 0.1% trifluoroacetic acid, and 5 mg mL −1 ammonium citrate. Dried samples were massed with MALDI-TOF on a Bruker Daltonics ultraflex II TOF/TOF and analyzed using flexAnalysis 3.4. Small unilamellar vesicle (SUVs) preparation SUVs were prepared using a 10 mL LIPEX Extruder (Transferra Nanosciences, Inc.). Lipids were mixed in ~500 μL chloroform with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (850375C, Avanti Polar Lipids) as the base lipid. Biotinylated lipids were incorporated at 0.05–0.2 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Biotinyl-Cap PE) (870282C, Avanti Polar Lipids). To directly tag the membrane in control experiments, N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (FITC DHPE) (23304, AAT Bioquest) was incorporated at 0.1 mol%. Lipids were dried first by rotary evaporation and second by ultrahigh purity N 2 . Lipid cakes were resuspended and sonicated in 3 mL nanopure water (final concentration, 2 mg mL −1 ) prior to performing three freeze-thaw cycles. SUVs in nanopure water were then extruded 10× through a 0.08 μm polycarbonate filter (WHA110604, Whatman) supported by a drain disc (WHA230600, Whatman). SUVs were used within ~2 weeks. SLB Preparation Planar SLBs were prepared on either uncoated glass-bottom 96-well plates (265300, Nunc or 82050-782, Greiner) or glass coverslips (48382-085, VWR). Coverslips were washed and sonicated 3× in nanopure water followed by sonication in ethanol. Coverslips were dried at 90 °C overnight and cleaned in piranha solution (3:1 sulfuric acid and 30% hydrogen peroxide; caution, piranha acid is extremely corrosive and can explode if exposed to organic materials). Cleaned coverslips were washed 3× in nanopure water and were mounted into coverslip chambers in 1× PBS for SLB formation. Alternatively, 96-well plates were etched for 1–3 h in 2.6 M sodium hydroxide and were washed with 10 mL nanopure water and 5 mL 1× PBS. SLBs were formed by adding SUVs to etched glass for at least 5 min and were washed in nanopure water and 1× PBS prior to ~25 min blocking with 0.1% bovine serum albumin, Fraction V (10 735 078 001, Roche Diagnostics GmbH). Unless otherwise stated, all experiments were carried out with 99.9 mol% DOPC and 0.1 mol% Biotinyl-Cap PE. Blocked SLBs were washed with 5 mL 1× PBS and then saturated in 90–180 nM streptavidin (SA101, Millipore-Sigma) for at least 45 min. Excess streptavidin was removed with 10 mL 1× PBS, and SLBs were incubated with 30 nM DNA for at least 45 min. Functionalized SLBs were washed in 10 mL 1× PBS and then buffer exchanged into hanks balanced salts (Millipore-Sigma) for all imaging. To stain the membrane, SLBs were shaken for 30 min at 240 rpm with 10% (v/v) 250 μg μL −1 b-BODIPY FL C 5 -HPC (2-(4,4-difluoro-5,7, dimethyl-4-bora-3a,4a-diaza- s -indacene-3-pentanoyl)-1-hexadecaonyl- sn -glycero-3-phosphocholine (D3803, Thermo Fisher) or were incubated with 10% (v/v) 1.5 μg mL −1 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DiD) (D7757, Thermo Fisher) (DID). For MFM bead experiments, SLBs were assembled on 5 μm silica beads (SS06N, Bang Laboratories). One hundred microliters of 1 mg mL −1 beads were rocked with 100 μL of DOPC SUVs. SLB-beads were washed in 1× PBS and purified 3× with centrifugation (5 min, 2000 rpm). Purified SLB-beads were incubated with 5 μM 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (468495, Thermo Fisher) for 15 min. Free dye was removed by washing and pelleting 3× in 1× PBS (5 min, 2000 rpm) 49 .

DNA hybridization

DNA oligonucleotides in 1× PBS were heated to 90 °C for 5 min and cooled at 25 °C for 25 min in a 0.2 mL thermowell tube. The ligand strand was added in 10% molar excess except for in absorbance spectroscopy and PC experiments, in which strands were added in a ratio of 1:1:1. To chemically open tension probes, tension probes were hybridized with 10× molar excess complementary sequence (Supplementary Figs. 6 and 8a ). UV-Vis spectroscopy Oligonucleotides (10 μL of 2.5 μM hairpin strand) were hybridized as described above to assemble closed tension probes in solution. Following hybridization, thermowell tubes were fit inside microcentrifuge tubes and dried in a vacufuge to 0.05, * P < 0.05, ** P < 0.01, *** P < 0.0001, **** P < 0.0001. Detailed information on statistical tests, reproducibility, and outlier omission are listed in each figure caption. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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