We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.
Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.
Mechanisms of how biomolecules respond to environmental stimuli have been studied widely. In a flow environment in particular, shear and elongational forces regulate the conformational changes and potentially the function of biomolecules. Typical examples include shear-induced unraveling of lambda DNA and von Willebrand factor (VWF). Lambda DNA has been used as a tool to understand conformational dynamics of individual, flexible polymer chains and the rheology of polymer solutions1,2,3,4. VWF is a natural flow sensor that aggregates platelets at wound sites of blood vessels with abnormal shear rates and flow patterns. Unraveling of VWF is essential in activating the binding of platelets to the A1 domain and collagen binding to the A3 domain. In addition, high shear-induced A2 domain unfolding allows the cleavage of VWF, which regulates its molecular weight distribution in circulation5,6. Thus, direct visualization of how these molecules behave under flow can greatly enhance our fundamental understanding of their biomechanics and function, which in turn can enable novel diagnostic and therapeutic applications.
Typical methodologies to characterize single-molecule conformations include optical/magnetic tweezers, atomic force microscopy (AFM) and single-molecule Förster resonance energy transfer (FRET)7. Single-molecule force spectroscopy is a powerful tool to investigate the force and motion associated with the conformational changes of biomolecules. However, it lacks the ability to map overall molecular conformations8. AFM is capable of imaging with high spatial resolution but is limited in temporal resolution9,10. In addition, contact between the tip and the sample may confound the response induced by flow. Other methods like FRET and nanopore analytics determine single-molecule protein folding and unfolding states based on the detection of intramolecular distance and excluded volumes. However, these methods are still in their infancy and limited in their direct observation of single-molecule conformations11,12,13,14.
On the other hand, directly observing macromolecules with high temporal and spatial resolution under fluorescence microscopy has improved our understanding of single-molecule dynamics in many biological processes15,16. For example, Fu et al. recently achieved simultaneous visualization of VWF elongation and platelet receptor binding for the first time. In their work, VWF molecules were immobilized on the surface of a microfluidic channel through biotin-streptavidin interactions and imaged under total internal reflection fluorescence (TIRF) microscopy at varying shear flow environments17. Applying a similar method as Fu's, we here demonstrate that conformations of VWF and lambda DNA can be directly observed under both TIRF and confocal fluorescence microscopy. As shown in Figure 1, microfluidic devices are used to create and control shear flow, and biomolecules are immobilized on the channel surface. Upon the application of varying shear rates, conformations of the same molecule are recorded to measure the extensional length, also shown in Figure 1. The method could be widely applied to explore other polymer behaviors under complex flow environments for both rheological and biological studies.
1. Preparing VWF
2. Preparing Lambda DNA
3. Creating microfluidic channel molds in silicon wafer
4. Preparation of polydimethylsiloxane (PDMS) microfluidic device
5. Treating surface of microfluidic device
6. Visualizing VWF and Lambda DNA under fluorescence microscopy
7. Image analysis of conformational changes
Observing the dynamic behavior of biomolecules such as VWF and lambda DNA is highly dependent on optimizing their binding to the device surface. Incubating surface treatments for the recommended times in the microfluidic device is crucial to obtaining binding with a few anchorage points, so that molecules can freely extend and relax upon changing flow. If the proteins or DNA are bound too strongly with multiple linkages, they will either extend to limited lengths or not extend at all. This occurs particularly with VWF when it remains without flow on the device surface for more than 3 min prior to free biotin blocking. The longer VWF remains on the surface stagnant, the more VWF biotin groups bind to the surface streptavidin groups and the less flexibility the molecule has to unravel. If molecules are bound too weakly, on the other hand, they will detach upon flow and disappear from view. This can occur if VWF or lambda DNA is incubated for too short of periods, causing too few biotin-streptavidin interactions to form. Molecules can also break free when extremely high shear rates (>200,000 s-1) are applied, weakening the biotin-streptavidin interactions.
An ideal molecule binds to such an extent that it can unravel and relax upon multiple cycles of stopping and starting flow. The flexibility of a molecule to change conformation like this is often demonstrated by its ability to extend to increasing lengths as higher shear rates are applied within a range of increasing flow. Images of VWF obtained with TIRF microscopy demonstrate this relationship in Video 1. The extension versus shear rate curve of this same VWF molecule in Figure 4 precisely captures the shear-induced behavior of a VWF molecule and is useful for characterizing the biomechanical properties of the protein. Images of lambda DNA obtained with confocal fluorescence microscopy similarly show increased extension upon higher shear rates and gradual relaxation over 2 min, as is captured in Video 2 and Video 3. The recoiling characteristics of lambda DNA after stopped flow is also graphically represented in Figure 5.
Figure 1: Schematics of single-molecule flow experiment in microfluidic channel under fluorescence microscopy. The channel surface is coated with BSA-biotin and blocked with casein. Streptavidin is bonded with biotin on the channel surface and also biotinylated VWF/lambda DNA to immobilize single molecules on the surface. As shear rate increases from A para C, the molecule is stretched from a folded state to an elongated state along the flow direction from the left to the right. Please click here to view a larger version of this figure.
Figure 2: Microfluidic channel dimensions. The shape and structure of the PDMS microfluidic device are shown together with the channel dimensions. The channel is 50 µm in height and ranges from 0.1 to 1.0 mm in width. The narrowing region in the middle of the channel is 0.7 mm in length. The inlet and the outlet are 0.5144 mm (25 G) in diameter. Flow direction is from the left to right. Please click here to view a larger version of this figure.
Figure 3: Surface treatment steps for single-molecule immobilization. All steps occur at room temperature. (A). BSA-biotin is coated on the surface for 2 h. (B). Casein is injected into the channel for 30 min to block the surface. (C). Streptavidin is incubated in the channel for 10 min to bind with BSA-biotin. (D). After washing away excess molecules in the former steps, fluorophore and biotin labeled VWF/lambda DNA is injected into the channel and immobilized through bonding with streptavidin. (E). Free biotin is flowed in, blocking extra streptavidin binding sites to minimize its interference with the molecule during conformational changes. Please click here to view a larger version of this figure.
Figure 4: Extensional behavior of VWF under shear flow. The molecule reversibly unravels at 7 different shear rates: 0 s-1, 33,333 s-1, 66,667 s-1, 100,000 s-1, 133,333 s-1, 166,667 s-1 and 200,000 s-1. Length of the stretched molecule increases from 0.52 µm at zero shear rate to 3.44 µm at 200,000 s-1 shear rate. Please click here to view a larger version of this figure.
Figure 5: Relaxation behavior of lambda DNA after shear flow stops. Flow with 33,000 s-1 and 66,667 s-1 shear rates are applied from 0 to 30 s to the same molecule. Relaxation is recorded from 30 s to 150 s. At 66,667 s-1 shear rate, the DNA molecule elongates to 15.00 µm and relaxes back to 5.83 µm after the flow has been stopped for 2 min. At 33,333 s-1 shear rate, the molecule extends only to 8.75 µm and is 3.33 µm in length after 2 min of relaxation. Please click here to view a larger version of this figure.
Video 1: Reversible unraveling of VWF under increasing shear rates using total internal reflection fluorescence (TIRF) microscopy. The molecule in the middle of the view reversibly unravels to different length at shear rates 33,333 s-1, 66,667 s-1, 100,000 s-1 and 133,333 s-1. A syringe pump is used to control the flow rate from which shear rates are calculated. Flow direction is from the left to right. Images are taken with 15 s intervals to allow complete relaxation and extension processes. Please click here to view this video. (Right-click to download.)
Video 2: Relaxation of lambda DNA after 33,333 s-1 shear rate. Images are taken under confocal fluorescence microscopy. Lambda DNA are stretched under 33,333 s-1 shear flow and relaxed back to a folded state after the flow is stopped at 30 s. Duration of the relaxation is 2 min. Flow direction is from the left to right. Images are taken with 30 s intervals in between. Please click here to view this video. (Right-click to download.)
Video 3: Relaxation of lambda DNA after 66,667 s-1 shear rate. Settings are identical to the ones in Video 2 except for the initial shear rate. Please click here to view this video. (Right-click to download.)
Supplementary Files: MATLAB codes. Please click here to download this file.
To obtain high quality data of single-molecule conformational changes using fluorescence microscopy as described in this method, it is critical to incubate the molecule for the appropriate amount of time, minimize its nonspecific interactions with the surface and establish microscope settings that reduce photobleaching. The ability of the molecule to freely change conformation is related to the number of biotin-streptavidin interactions formed between the molecule and the surface. As mentioned previously, this must be controlled by incubating the molecule without flow for the appropriate amount of time. Additionally, protein or DNA may nonspecifically bind to the coverslip if the coverslip is not blocked effectively. Without the recommended blocking solution, molecules can attach to the glass nonspecifically and be unresponsive to any flow rate applied. Applying the casein block during early surface treatment and maintaining its presence during flow is essential for reducing these nonspecific interactions. Finally, capturing the continuous, dynamic behavior of a single molecule requires frequent fluorophore excitement during image capture. This can cause rapid photobleaching if laser intensity, exposure time and exposure frequency are too high. It is therefore necessary to adjust these settings in tandem and strategize how to reduce their values without compromising the time or image resolution of the data.
If extension and relaxation of the molecule are not observed, additional steps should be followed. Incubate the molecule in the device for longer and shorter times than what is advised in the protocol. For each time that is tested, vary BSA-biotin and streptavidin concentrations by factors of 10. These tests may be necessary to optimize the number of biotin-streptavidin anchorage points formed between the molecule and surface. For example, if the biotin labeling density is very high, due to deviations from the recommended concentrations or reagents in the labeling protocol, shorter molecular incubation time and lower BSA-biotin and streptavidin concentrations may be needed. To further improve the success of the experiment, scan the entire microfluidic device for molecules that reversibly unravel. The surface may not be treated uniformly with streptavidin or casein block, causing molecules in certain areas to have greater unraveling responses than others.
This method is limited by a lack of information about the size and tethering points of the molecule, the difficulty in producing 0 s-1 shear rate and the optical resolution of fluorescence microscopes. Previous work has shown a large variation in the unraveling behavior of VWF, potentially explained by the wide distribution in the number and location of biotin-streptavidin tether points and the molecular weight of each VWF molecule18. At the moment, the method we present cannot define tether points and molecular size. However, Brownian dynamics simulations of a coarse-grained VWF model published by Wang et al. incorporate these variables and can be run alongside experimental findings to explain such variation18. Furthermore, flow does not stop instantaneously when the syringe pump is stopped, confounding the observation of recoiling dynamics. This is due to the deformation and slight dilation of the PDMS channel during the intended flow period. When the pump is stopped, fluid continues to flow until the PDMS is fully relaxed. An improved system should use more rigid PDMS or microchannels fabricated in hard plastic materials, allowing fluid to reach a 0 s-1 shear rate more quickly. Finally, one can only resolve molecules whose size is on the same order of magnitude as the optical resolution of the fluorescence microscope, which may be no smaller than a few hundred nanometers. Thus, there is a minimum size requirement for the molecules that can be directly observed with this method.
The current protocol concerns mainly quantification of conformational changes of protein and DNA molecules under physiological flow. However, the method can also be used to visualize real-time interactions between biological molecules and further characterize protein and DNA function. For instance, Fu et al. have shown that tethered VWF can activate under high shear flow and further capture the platelet adhesion molecule GPIbα under varying flow conditions17. This binding event is preserved even when VWF is bound to the surface by biotin-streptavidin linkages, demonstrating the effectiveness of this protocol to study physiologically relevant functions and mechanics17. Similar mechanistic insights could be obtained while studying the interactions between unraveled DNA and regulatory proteins in flow environments21,22. Additionally, our method pertains mostly to observing conformational changes in macromolecules. Nevertheless, one could adapt it for the purpose of studying smaller molecules that are large enough to be resolved under fluorescence microscopy. For example, by noncovalently or covalently attaching a small molecule to a much larger, immobilized lambda DNA, one could increase the shear-sensitivity of the smaller molecule and more easily observe its behavior. In conclusion, other single-molecule characterization methods, such as AFM or optical tweezers, provide high-resolution data on the structural and functional properties of macromolecules; however, these alternative methods cannot observe the dynamic, conformational changes of proteins and DNA that take place in a physiological flow environment, as is presented in this protocol.
The authors have nothing to disclose.
This work was supported in part by a National Science Foundation grant DMS-1463234, National Institutes of Health grants HL082808 and AI133634, and Lehigh University internal funding.
Alexa Fluor 488 Labeling Kit | Invitrogen | A30006 | |
Bio-Spin P-6 Gel Columns | Bio-Rad | 7326221 | |
Biotin | Sigma-Aldrich | B4501 | Use as free biotin in Step 5.6 |
Biotin-14-dCTP | AAT Bioquest | 17019 | |
BSA-Biotin | Sigma-Aldrich | A8549 | |
Coverslips | VWR | 48393-195 | No. 1 ½, 22 x 50 mm |
dNTP Set | Invitrogen | 10297018 | |
Float Buoys for Mini Dialysis Device | Thermo Scientific | 69588 | |
Klenow Fragment (3'→5' exo-) | New England BioLabs | M0212S | Use for 10X reaction buffer in Step 2.1.1 and 1X reaction buffer in Step 2.2.2 |
Lambda DNA | New England BioLabs | N3011S | |
Mini Dialysis Device | Thermo Scientific | 69570 | 10K MWCO, 0.1 mL volume |
NEBuffer 4 | New England BioLabs | B7004S | |
NHS-PEG4-Biotin | Thermo Scientific | 21330 | |
Protocatechuate 3,4-Dioxygenase | Sigma-Aldrich | P8279 | |
Protocatechuic acid | Santa Cruz Biotechnology | sc-205818 | |
Silicone Elastomer Kit for PDMS Fabrication | The Dow Chemical Company | 4019862 | |
Streptavidin | Sigma-Aldrich | 85878 | |
The Blocking Solution | CANDOR Bioscience | 110 050 | Use as casein blocking solution throughout protocol |
Vinyl Cleanroom Tape | Fisher Scientific | 19-120-3217 | |
von Willebrand Factor, Human Plasma | Millipore Sigma | 681300 | |
YOYO-1 Dye | AAT Bioquest | 17580 | |
0.25 mm Inner Diameter Tubing | Cole-Parmer | EW-06419-00 | |
25 Gauge Needle | Thomas Scientific | JG2505X |