This research work was supported by the National Science Foundation [CBET-1904921] and the National Institutes of Health [NIH R01CA236350] to H. M.

NOTE: All the buffers and the chemical reagents used in this protocol are listed in the Table Materials.
1. Preparation of the shear force microscope
NOTE: The shear force microscope contains two parts, a reaction unit (homogenizer) and a detection unit (fluorescence microscope). The magnification of the eyepiece is 10x, and the magnification of the objective lens (air) is 4x.
<ol>
	<li>Assemble the homogenizer and the microscope ...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+assay+for+proteolytic+susceptibility:+Force-induced+collagen+destabilization.">Single-molecule assay for proteolytic susceptibility: Force-induced collagen destabilization.</a> Biophysical Journal. 114 (3), 570-576 (2018).</li><li>Astumian, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+and+kinetics+of+molecular+motors.">Thermodynamics and kinetics of molecular motors.</a> Biophysical Journal. 98 (11), 2401-2409 (2010).</li><li>Bekard, I. B., Asimakis, P., Bertolini, J., Dunstan, D. 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The protocol described in this manuscript allows real-time investigation of the unfolding of an ensemble set of biomolecular structures by shear force. The results presented here underscore that DNA i-motif structures can be unfolded by shear force. The unfolding of the ligand-bound i-motif and the shear force-actuated click reactions were proof-of-concept applications for this ensemble force spectroscopy method.
Figure 1 presents the instrument setup. The homogen...

In single-molecule force spectroscopy1 (SMFS), the mechanical properties of individual molecular structures have been studied by sophisticated instruments such as the atomic force microscope, optical tweezers, and magnetic tweezers2,3,4. Restricted by the same directionality requirement of the molecules in the force-generating/detecting setups or the small field of view in magnetic tweezers and the miniature centrifuge force microscope (MCF)5,6,7,8, only a limited number of molecules can be investigated simultaneously using SMFS. The low throughput of SMFS prevents its wide application in the molecular recognition field, which requires the involvement of a large set of molecules.
Shear flow provides a potential solution to apply forces to a massive set of molecules9. In a liquid flow inside a channel, the closer to the channel surface, the slower the flow rate10. Such a flow velocity gradient causes shear stress that is parallel to the boundary surface. When a molecule is placed in this shear flow, the molecule reorients itself so that its long axis aligns with the flow direction, as the shear force is applied to the long axis11. As a result of this reorientation, all the molecules of the same type (size and length of handles) are expected to align in the same direction while experiencing the same shear force.
This work describes a protocol to use such a shear flow to exert shear force on a massive set of molecular structures, as exemplified by the DNA i-motif. In this protocol, a shear flow is generated between the rotor and stator in a homogenizer tip. The present study found that the folded DNA i-motif structure could be unfolded by shear rates of 9724-97245 s&#8722;1. Besides, a dissociation constant of 36 &#181;M was found between the L2H2-4OTD ligand and the i-motif. This value is consistent with that of 31 &#181;M measured by the gel shift assay12. Further, the current technique is used to unfold the i-motif, which can expose the chelated copper (I) to catalyze a click reaction. This protocol thus allows one to unfold a large set of i-motif structures with low-cost instruments in a reasonable time (shorter than 30 min). Given that the shear force technique drastically increases the throughput of the force spectroscopy, we call this technique ensemble force spectroscopy (EFS). This protocol aims to provide experimental guidelines to facilitate the application of this shear force-based EFS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3K MWCO Amicon</td>
			<td>Millipore Sigma</td>
			<td>ufc900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>VWR</td>
			<td>VWRC0143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calfluor 488 azide</td>
			<td>Click Chemistry Tools</td>
			<td>1369-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl</td>
			<td>Thermo&#160;</td>
			<td>ACRO270525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispersion tip</td>
			<td>Switzerland</td>
			<td>PT-DA07/2EC-B101</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligos</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dye</td>
			<td>IDT</td>
			<td>/5Cy5/</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Janpan</td>
			<td>Nikon TE2000-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Switzerland</td>
			<td>PT 3100D</td>
			<td></td>
		</tr>
		<tr>
			<td>HPG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>cs-295271</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26760.295</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>VWR</td>
			<td>VWRCE169-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quencher</td>
			<td>IDT</td>
			<td>/3IAbRQSp/</td>
			<td></td>
		</tr>
		<tr>
			<td>TBTA</td>
			<td>Tokyo Chemical Industry</td>
			<td>T2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>VWRCE133-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

ensemble force spectroscopy by shear forces

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the lack of high throughput capabilities, the application of these techniques is limited in biophysics. Ensemble force spectroscopy (EFS) has demonstrated high throughput in the investigation of a massive set of molecular structures by converting mechanochemical studies of individual molecules into those of molecular ensembles. In this protocol, the DNA secondary structures (i-motifs) were unfolded in the shear flow between the rotor and stator of a homogenizer tip at shear rates up to 77796/s. The effects of flow rates and molecular sizes on the shear forces experienced by the i-motif were demonstrated. The EFS technique also revealed the binding affinity between DNA i-motifs and ligands. Furthermore, we have demonstrated a click chemistry reaction that can be actuated by shear force (i.e., mechano-click chemistry). These results establish the effectiveness of using shear force to control the conformation of molecular structures.

Figure 1 outlines the mechanical unfolding and real-time sensing of ensemble molecules in EFS. In Figure 1B, the fluorescence intensity of i-motif DNA was observed to increase with the shear rate ranging from 9,724 s&#8722;1 to 97,245 s&#8722;1 in a pH 5.5 MES buffer. As a control, fluorescence intensity was not increased when the same i-motif DNA was sheared at a rate of 63,209 s&#8722;1 in a pH 7.4 MES buffer. ...

Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Ensemble Force Spectroscopy by Shear Forces

Ensemble force spectroscopy (EFS) is a robust technique for mechanical unfolding and real-time sensing of an ensemble set of biomolecular structures in biophysical and biosensing fields.

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

This protocol provides a high throughput technique to study the intermolecular binding between DNA and ligands. This technique has demonstrated high throughput in the investigation of massive set of biomolecular structures. It does so by converting the mechanical chemical properties of individual molecules into those of molecular ensembles.I think it is a very step forward experiment but there are few critically steps. For example, the alignment of microscope, homogenizer, and the reaction tube. These three things should be perfectly aligned in order to accurately track the change in fluorescence.Begin by assembling the homogenizer and the microscope on a mounting table. After wearing goggles, turn on the fluorescence microscope and then adjust the homogenizer to make sure that the excitation light beam passes through the center of the dispersing tip of the homogenizer. Then prepare a flat bottomed reaction chamber that is five centimeters in height and 1.5 by 1.5 square centimeters in cross section, ensuring that the selected chamber material does not fluoresce to reduce the background.Afterward, clamp the reaction chamber on the sample stage of the fluorescence microscope and then adjust the chamber to hold the dispersing tip of the homogenizer, ensuring that the tip is slightly above the bottom surface of the reaction chamber. Next, adjust the vertical position of the homogenizer and the chamber together to ensure the focus of the microscope is on the surface of the dispersing tip. Then adjust the horizontal position of the dispersing tip to ensure that the detection area is set between the rotor and stater.Switch on the fluorescent channels, according to the fluorescent dye used in the experiment. Before the high speed shearing experiment, used deionized water to test the shearing with a low shearing speed. First, prepare a human telomeric i-motif DNA labeled with a dye and a quencher at its two ends respectively in deionized water as described in the manuscript.Next, dilute the DNA to five micromolar in 30 millimolar MES buffer at pH 5.5 or pH 7.4. To the DNA solution, add ligand L2H24OTD"in a concentration range of zero to 60 micromolar and mix the solution gently for 10 minutes to fold the i-motif structures without light. Check and minimize the background fluorescence intensity of the reaction chamber that is filled with deionized water using the fluorescent microscope without shearing.Then set the parameters of the CCD camera using the software with exposure time as 0.5 seconds, CCD sensitivity of 1, 600, and recording time equal to 20 minutes. Using a long pipette, add the DNA solution into the empty and clean reaction chamber and cover the reaction chamber with a black box. Then start the homogenizer to perform shearing at a selected shear rate ranging from 9, 724 per second to 97, 245 per second for 20 minutes, with the CCD camera turned on to record the data.After the experiment, remove the chamber and wash it with deionized water. Prepare i-motif DNA and deionized water. Then, incubate 10 micromolar i-motif DNA and 300 microliters of 30 millimolar tris buffer supplemented with 150 micromolar cupric chloride and 300 micromolar ascorbic acid for 10 minutes to fold the i-motif structures.Next ultra filtrate the solution with an ultra filtration device at a centrifugal force of 14, 300 times G.Replenish the solution to approximately 500 microliters with 30 millimolar tris buffers supplemented with 300 micromolar ascorbic acid after each filtration. After collecting the residual solution, make a final volume of 300 microliters by adding 30 millimolar tris supplemented with 300 micromolar ascorbic acid along with 20 micromolar CalFluor 488 Azide, 20 micromolar HPG, and 10 micromolar TBTA. Once the reagents are added, move the solution to the dark room.Then check and minimize the background fluorescence intensity of the reaction chamber filled with deionized water using the microscope before the shearing experiments. Next, add the DNA solution into the empty reaction chamber with a long pipette, and then start the homogenizer shearing at a shear rate of 63, 209 per second for 20 minutes with the CCD camera turned on. After the experiment, remove the chamber and wash it with deionized water.The fluorescence intensity of i-motif DNA was observed to increase with the sheer rate ranging from 9, 724 per second to 97, 245 per second in a pH 5.5 MES buffer. However, fluorescence intensity was not increased when the same i-motif DNA was sheared at a rate of 63, 209 per second, as it does not fold at pH 7.4 in MES buffer. The ligand binding to the DNA i-motif molecules subjected to the sheer flow was evaluated which showed that the increment of the fluorescence intensity became less obvious with increasing ligand concentration.The dissociation constant was determined for the interaction between the ligand and the i-motif and was found to be 34 micromoles. The copper one chelated i-motif was unfolded at the 63, 209 per second shear rate, and the released copper one triggered the fluorgenic click reaction, resulting in increased fluorescence intensity over time. Firstly, make sure that the detection area is set between rotor and the stater.Second, check and minimize the fluorescence background. And lastly, make sure you filter the reaction before you perform click reaction. This method opens a gateway to explore mechanical strength, folding and unfolding off all kinds of polymers.It could be other DNA structures, proteins, or other polymers, and their interaction with other kinds of molecules. As said, several other macromolecules could be studied using this approach.

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1.4K vistas.  Kent State University. Este protocolo proporciona una técnica de alto rendimiento para estudiar la unión intermolecular entre el ADN y los ligandos. Esta técnica ha demostrado un alto rendimiento en la investigación de un conjunto masivo de estructuras biomoleculares. Lo hace convirtiendo las propiedades químicas mecánicas de moléculas individuales en las de conjuntos moleculares.Creo que es un experimento muy avanzado, pero hay pocos pasos críticos. Por ejemplo, la alineación del microscopio, el ho...