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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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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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.
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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 (ÎĽx and ÎĽy) 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 ÎĽM of 24nt-10comp). Frequent anti-correlated dynamics in diffusion coefficient (D) and electrokinetic mobility (ÎĽx and ÎĽy) 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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DOI: https://doi.org/10.1038/nmeth.2882
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