The overall structure of the DNA probe comprises two DNA fragments assembled by base pairing. One is the main hairpin structure of 74 nt in length with an overhang at its 3' end, which we name here as the Hairpin Strand 74 (HS74), and the other is a single DNA strand of 21 nt with a black hole quencher at its 3' end anchoring on the overhang of the hairpin structure. Three functional groups are modified on HS74: 5' end fluorophore, intermediate cyclic peptide RGDfK, and 3' end S-S bond. In order to obtain HS74, we first synthesized two oligonucleotides “bricks” (strand I and II), where strand I consists of a Cy3B dye at the 5'-end and a cyclic RGD peptide (cRGDfK) at an internal position and strand II presents a phosphoric acid at the 5'-terminal and a thiol group at the 3'-end ((Figure 1 a and b). We then linked them by DNA ligase. All the sequences of oligonucleotides are list in the Reagents Part. The following is the detailed experimental procedure (Figure 1c).
Conjugate the Cy3B-maleimide to the thiol group on the strand I (40 nt)
1. Treat thiol modified strand I with Tris(2-carboxyethyl) phosphine (TCEP) solution (10 mM TCEP, 100 mM sodium phosphate,150 mM sodium chloride, pH 7.2) for 2 h at room temperature to reduce the partially formed disulfide bond, followed by passing through a Micro Bio-Spin P-6 Gel Column (Bio-Rad, 7326221) equilibrated with conjugation buffer (100 mM sodium phosphate,150 mM sodium chloride, 5 mM EDTA, pH 7.0). DNA concentration can be determined by using Nanodrop UV-Vis Spectrophotometer.
2. Add 5-fold molar excess of Cy3B-maleimide dissolved in DMSO over the amount of strand I-thiol into strand I solution. Incubate the reaction buffer for 2 hours at room temperature.
3. Separate the conjugate (DNA-Cy3B) from the excess Cy3B and by-products by passing through a Micro Bio-Spin P-6 Gel Column equilibrated with ultra-pure water.
4. An AQ-C18 column (diameter: 4.6 mm; length: 250 mm) in a reverse phase binary pump HPLC is used for further purification of the DNA product (flow rate 1 mL/min; solvent A: 0.1M TEAA, solvent B: 100% acetonitrile; initial condition was 10% B; gradient: 1% B per min), and then the separated product can be dried by rotation evaporation.
Label DNA strand I with a cyclic peptide RGDfK (cRGDfK)
Prepare the reaction solution as this: 50% DMSO; 0.5 mM Ascorbic acid; 0.5 mM Cu-TBTA complex; alkyne modified DNA strand I-Cy3B; 1.5-fold Azide-cRGDfK; 0.2M triethylammonium acetate buffer, pH 7.0.
1. Vortex the mixture thoroughly and then keeps it at room temperature overnight.
2. Separate the Cy3B-oligonucleotide-cRGDfK conjugate from unmodified oligonucleotide by using denaturing polyacrylamide gel electrophoresis (PAGE) (employing 8M urea).
3. Cut the desired band from the gel and mince. The product can be extracted in PBS at 4 °C overnight, subsequently followed by filtration through a 0.45 um filter tube to remove the gel debris. Alternatively, the extraction can be performed on a vortex device, and this will promote the diffusion of oligonucleotide out of the gel.
4. Desalt and purify the conjugation by using a Micro Bio-Spin P-6 Gel Column equilibrated with PBS.
Ligate labeled strand I and strand II
1. Mix the labeled strand I, strand II and a template strand which is partially complementary to strand I-strand II conjugate in a molar ratio of 1.1:1.1:1. The mixture is then heated to 94 °C for 5 min followed by naturally cooling to room temperature for annealing.
2. After annealing, using T4 DNA ligase to ligate strand I and strand II referring to the product manual.
3. Further purify the ligation product of 74 nt by using denaturing PAGE. And the following steps (extraction and desalting) are the same as described above.
Assemble the quencher-strand III with HS74
Mix strand III and HS74 in a molar ratio of 1.1:1. The mixture is then heated to 94 °C for 5 min and followed by naturally cooling to room temperature for annealing. And up to this step, the tension sensor can be ready for use.
Functionalization of glass coverslips with tension sensors
1. Sonicate the glass coverslip (25-mm in diameter) in ultrapure water and ethanol 3 times for 10 min each.
2. Dry the coverslip at 80 °C; etch either in piranha (a 3:1 mixture of sulfuric acid and hydrogen peroxide) for 40 min or in oxygen plasma (gas pressure: 200 mTorr) for 10 min can be powerful to build hydroxylated Surface.
3. Immerse the hydroxylated coverslip into 1% (3-aminopropyl) triethoxysilane (APTES) in ethanol and incubate for 1 h at room temperature.
4. After amination, discard the solution and add fresh ethanol. Then rinse the coverslip with ethanol and dry the coverslip under nitrogen.
Note a: in order to completely remove the physically adsorbed APTES, the coverslip needs to be rinsed thoroughly and sonication in fresh ethanol is a powerful means.
Note b: a curing step at 80 °C for 1 h is always chose following APTES deposition, meant to form stable covalent bond between APTES. But we find no additional improvement in surface quality after curing.
5. After silanization, PEGylate the coverslip with 5% PEG and 0.5% lipoic acid PEG. Briefly, first prepare the PEGylation solution:5% PEG NHS ester and 0.5% lipoic acid PEG NHS ester in 0.1M sodium bicarbonate, pH 8.5, then drop 100 ul of this solution to the amine-modified surface of the coverslip and cover the drop with another coverslip carefully, making sure that both the amine-modified surfaces of coverslips face the PEG solution, leaving it at 4 °C overnight or at room temperature for 2 h.
6. After the coverslip is thoroughly rinsed with ultrapure water, add 14 nM Au nanoparticles (AuNPs) 5-nm in diameter onto the coverslip and incubate for 30 min. Then rinse the coverslip with ultrapure water, leaving the fixed AuNPs on the surface which is strongly combined by Au-S bond between AuNP and the lipoic acid group on the surface.
7. Deposit 5 ul drops of sensor solution (50 nM force sensor, 1M NaCl, 20 mM sodium phosphate, pH 7.2) onto the places of AuNP, and incubate for more than 1h; after washed with PBS, then the sensor functioned coverslip will be ready for seeding cells imaging under a total internal reflection microscope (TIRF).
NIH 3T3 cells are cultured in DMEM medium with high glucose supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G, and 100 µg/mL streptomycin.
1. Detach the cells by using pre-warmed trypsin/EDTA solution.
2. Once the cells appear detached, add 2 volumes of complete medium to inactivate trypsin. Disperse the cells by gently pipetting over the cell layer surface and then transfer the cells to the tube, followed by centrifuge at100-g for 5 min. After removing the supernatant, gently resuspend the cell with a complete growth medium. Pipet a drop of the cell solution to a petri dish and check the density of the cells under microscopy.
3. Assemble the force sensor functioned coverslip into the coverslip cell chamber for live-cell microscopy.
4. Pipet appropriate volume of the cell solution to the cell chamber, and incubate the cells in a incubator (37 °C, 5% CO2).