Name: ensemble force spectroscopy by shear forces
Uploaded: 2022-07-26T13:00:00
Description: Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

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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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 ...

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