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Single-molecule motions enable direct visualization of biomolecular interactions in solution

Abstract

Biomolecular interactions are generally accompanied by modifications in size and charge of biomolecules at the nanometer scale. Here we describe a single-molecule method to sense these changes in real time based on statistical learning of diffusive and electric field–induced motion parameters of a trapped molecule in solution. We demonstrate the approach by resolving a monomer-trimer mixture along a protein dissociation pathway and visualizing the binding-unbinding kinetics of a single DNA molecule.

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Figure 1: Measurement principle and transport-based single-molecule identification.
Figure 2: Resolving monomers and trimers of APC in solution along the dissociation pathway.
Figure 3: DNA hybridization kinetics visualized by dynamic changes in transport coefficients in solution.

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Acknowledgements

We thank Y. Jiang for help with high-performance liquid chromatography purification, and C. Calderon, G. Schlau-Cohen, H.-Y. Yang, S. Bockenhauer and S.J. Sahl for discussion. This work is funded in part by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the US Department of Energy through grant DE-FG02-07ER15892.

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Authors

Contributions

Q.W. and W.E.M. conceived the project, discussed the results and wrote the manuscript. Q.W. designed and performed the experiments and data analysis.

Corresponding author

Correspondence to W E Moerner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–19, Supplementary Tables 1–5 and Supplementary Notes 1–7 (PDF 7165 kb)

Real-time estimation of single-molecule transport coefficients in an anti-Brownian electrokinetic trap.

Screen recording during an experiment trapping single Alexa647 labeled 10nt-ssDNA molecules without complementary strand. Intensity (I) is photon counts every 10 ms, diffusion coefficient (D) and electrokinetic mobility (μx and μy) are estimated every 5,000 photons using the real-time EM algorithm. Occasional intensity spikes are indicative of transient co-occupancy of two objects in the trap. Time axis units: 10 ms (AVI 4611 kb)

Real-time visualization of single-DNA binding-unbinding dynamics.

Screen recording during an experiment trapping single 10nt-ssDNA in presence of unlabeled complementary strand (2 μM of 24nt-10comp). Frequent anti-correlated dynamics in diffusion coefficient (D) and electrokinetic mobility (μx and μy) visualize transitions between ssDNA (blue band) and dsDNA (red band). Time axis units: 10 ms (AVI 10418 kb)

Supplementary Software

C program implementation of the parameter estimation algorithm. (TXT 17 kb)

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Wang, Q., Moerner, W. Single-molecule motions enable direct visualization of biomolecular interactions in solution. Nat Methods 11, 555–558 (2014). https://doi.org/10.1038/nmeth.2882

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