SINGLE-MOLECULE SENSING AND CHARACTERIZATION OF NATIVE, UNLABELED PROTEINS USING SOLID-STATE NANOPORES

Nanopores (NPs) are single molecule biosensors, which utilize electrokinetic focusing and transport to detect and characterize unlabeled biomolecules.¹ Controlling and tuning the capture rate and the translocation speed of biomolecules through the NP are crucial features to allow sensing of fine biomolecular features within the experimental bandwidth limitations. We recently developed methods to enhance the resolution of native, unlabeled proteins sensing by radically enhancing their residence time while in the pore, and improved nano-sensor morphology.² With these improvements we are able to sense and characterize individual native (chemically unmodified) proteins. Specifically, we demonstrate the discrimination among two, unlabeled, Ubiquitin (Ub) protein dimers, which only possess a slightly different 3D structure and having the same amino-acid sequence.³ Furthermore we are able to directly sense and map individual transcription-factors bound to their target ds-DNA.⁴ The ability to analyze biomolecular complexes, fine features along DNA strands and small proteins in their native folded state at the single molecule level opens up broad opportunities in biomedical research and in biochemistry.

References

1. Wanunu, M. & Meller, A. in Laboratory Manual on Single Molecules (eds. Selvin, P. R. & Ha, T.) Cold Spring Harbor Press (2008).
2. Di Fiori N, Squires A, Bar D, Gilboa T, Moustakas TD, Meller A. Optoelectronic control of surface charge and translocation dynamics in solid-state nanopores. Nature Nanotechnol 8, 946–951 (2013).
3. Nir, I., Huttner, D. & Meller, A. Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores. Biophys. J. 108, 2340–2349 (2015).
4. Squires, A., Atas, E. & Meller, A. Nanopore sensing of individual transcription factors bound to DNA. Sci. Rep. 5, 1–11 (2015).