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