Ensemble Force Spectroscopy by Shear Forces

Summary

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.

Abstract

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.

Introduction

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 tweezers^2,3,4. 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,

Protocol

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.

1. Assemble the homogenizer and the microscope on a mounting table. Wear goggles and turn on the fluorescence microscope, and then adjust the homogenizer to make sure that the excitation light beam....

Results

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⁻¹ to 97,245 s⁻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⁻¹ in a pH 7.4 MES buffer. .......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
3K MWCO Amicon	Millipore Sigma	ufc900324
Ascorbic acid	VWR	VWRC0143-100G
Calfluor 488 azide	Click Chemistry Tools	1369-1
CuCl	Thermo	ACRO270525000
Dispersion tip	Switzerland	PT-DA07/2EC-B101
DNA oligos	IDT
Dye	IDT	/5Cy5/
Fluorescence microscope	Janpan	Nikon TE2000-U
Homogenizer	Switzerland	PT 3100D
HPG	Santa Cruz Biotechnology	cs-295271
KCl	VWR	VWRC26760.295
MES	VWR	VWRCE169-500G
Quencher	IDT	/3IAbRQSp/
TBTA	Tokyo Chemical Industry	T2993
Tris	VWR	VWRCE133-100G