A Reversible shearing DNA-based tension probe for cellular force measurement

Cells can sense and respond to molecular forces ranging from a few pN to tens of pN through mechanosensitive receptors with an astounding diversity of mechanisms. DNA-based molecular tension sensors have been instrumental in studying the importance of mechanical forces in many biological systems. However, the respective shortcomings of these sensors, for instance, the irreversible rupture of tension gauge tether (TGT) under force and relatively limited dynamic range of the hairpin probes, limited our understanding of the molecular details of mechano-chemo-transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular pN-scale forces between 4-60 pN transmitted by cells. Using RSDTPs to study integrin-mediated mechanotransduction, we could real-time distinguish the differences of force-bearing integrins without perturbing adhesion biology in living cells.


Introduction
Cells sense various mechanical cues from the extracellular matrix (ECM) and their neighbors throughout the life-cycle, and their functional responses to these signals after properly transduced are vital to critical biological processes, including embryogenesis and immunity 1 .Major efforts have therefore been made to understand the relevant signaling pathways in mechanobiology, and scientists have discovered several key components of the underlying molecular machinery, of which mechanical force has emerged as a central player in general functions like adhesion and migration.The mechanical forces generated in these processes are tiny, in the range of a few pN to tens of pN on individual receptors 2 , but these forces can precisely regulate the signal transduction process in time and space, directly or indirectly controlling a number of biological responses such as cell differentiation, gene expression, and apoptosis 3 .
Over the last decade, various types of immobilized molecular uorescent tension probes have been developed to quantitatively measure the mechanical force exerted by speci c membrane proteins with pN sensitivity in living cells [4][5][6][7][8][9][10][11] .Among these probes, DNA-based probes have been widely used due to their easy-to-use synthesis procedure and programmability 6,7,10,11 .For instance, DNA hairpin-based tension probe with a range of 1-21 pN can successfully measure the activation forces from integrins 6,7 , T-cell receptor (TCR) 12 , and podosome structures 13 .Albeit successful, the mechanical ranges of the DNA hairpin-based tension sensors are not always su cient to cover the forces transduced on membrane receptors 8,14 .Recently, Wang and Ha constructed another type of DNA-based force sensor, called tension gauge tether (TGT) 11 , which relies on the irreversible force-dependent rupture of a DNA duplex to control and measure the cell adhesion forces.The rupture forces of TGT probe can be ne-tuned by changing the position of its mechanical loading site, thereby covering a wide range of tension (12-60 pN).Nevertheless, the rupture of TGT inevitability leads to forced breakages of receptor-ligand interactions, which could potentially disturb downstream signaling cascades and thus limit its applications.
Here, on the basis of the aforementioned DNA hairpin-based tension probes and rupture DNA duplexbased TGT probe, we developed a class of reversible shearing DNA-based tension probes (RSDTPs).By designing a new type of hairpin structure in which the rupture force can be adjusted either by varying the GC content in unzipping geometry, as in the conventional hairpin structure 6,7 or by tuning the positions of the mechanical loading sites in a shearing geometry, similar to the TGT probes 11 .Thus, the mechanical measurement of reversible shearing DNA probe can cover the 4-60 pN range.More importantly, RSDTPs presented here are non-rupturable that allow us to detect different ranges of integrin tension in real time.With these unique advantages, we believe RSDTPs could further explore mechano-signaling pathways behind the interplay between forces and biological processes.

Procedure
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 modi ed on HS74: 5' end uorophore, intermediate cyclic peptide RGDfK, and 3' end S-S bond.In order to obtain HS74, we rst 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 modi ed 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 disul de 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 puri cation of the DNA product ( ow 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.
1. Vortex the mixture thoroughly and then keeps it at room temperature overnight.
3. Cut the desired band from the gel and mince.The product can be extracted in PBS at 4 °C overnight, subsequently followed by ltration through a 0.45 um lter 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 Istrand 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 nd no additional improvement in surface quality after curing. 5.After silanization, PEGylate the coverslip with 5% PEG and 0.5% lipoic acid PEG.Brie y, rst 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-modi ed surface of the coverslip and cover the drop with another coverslip carefully, making sure that both the amine-modi ed 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) 5nm in diameter onto the coverslip and incubate for 30 min.Then rinse the coverslip with ultrapure water, leaving the xed AuNPs on the surface which is strongly combined by Au-S bond between AuNP and the lipoic acid group on the surface.the surface and were pulled directly off from the surface by cellular forces.This problem can be solved by increasing the concentration of lipoic acid-NHS ester and adding an additional amount of DMSO to promote the dissolution of lipoic acid-PEG.

Time Taken
It takes about 3 days for the synthesis of the DNA tension probe.Day 1: conjugate the Cy3B-maleimide to the thiol group on the strand I. Day 2: separate Cy3B-strand I through HPLC and perform the Click Chemistry conjugation.Day 3: ligate these two fragments (modi ed strand I and strand II) by using DNA T4 ligase followed by separation and puri cation of the product.

Anticipated Results
After the cells start spreading on the coverslip, we anticipate to observe clear integrin tension signals using total-internal-re ection uorescence (TIRF) microscopy (Figure 2).

Figures
Figures

Figure 1 Design
Figure 1