Nanopore sequencing is one of only a few methods that can potentially determine the amino acid sequence of individual protein molecules as these are passed through a pore sensor. However, mechanisms for unfolding and translocation of proteins are still unavailable to date. Here we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We combine a chemically resistant biological nanopore platform with a high concentration guanidinium chloride buffer to achieve unidirectional, single-file protein transport that is propelled by a giant electro-osmotic effect, as revealed by molecular dynamics simulations and confirmed experimentally. Remarkably, we observed that protein velocities are uniform regardless of the protein sequence, which allows the identification and discrimination among proteins based on their electrical signatures, as well as to distinguish protein signatures by their threading orientation (N-to-C vs. C-to-N terminus). With average transport velocities of 10 µs per amino acid, our method can enable direct, enzyme-free protein fingerprinting and protein sequencing when combined with a high-resolution pore and high-speed nanopore readout.
Use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leakfree at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers which exhibit deleterious leakage under similar conditions. Further, insertion and functional analysis of channels such as a-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that a-hemolysin channels maintain their structural integrity at 2M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.
In recent years, nanopore-based sequencers
have become robust tools
with unique advantages for genomics applications. However, progress
toward applying nanopores as highly sensitive, quantitative diagnostic
tools has been impeded by several challenges. One major limitation
is the insufficient sensitivity of nanopores in detecting disease
biomarkers, which are typically present at pM or lower concentrations
in biological fluids, while a second limitation is the general absence
of unique nanopore signals for different analytes. To bridge this
gap, we have developed a strategy for nanopore-based biomarker detection
that utilizes immunocapture, isothermal rolling circle amplification,
and sequence-specific fragmentation of the product to release multiple
DNA reporter molecules for nanopore detection. These DNA fragment
reporters produce sets of nanopore signals that form distinctive fingerprints,
or clusters. This fingerprint signature therefore allows the identification
and quantification of biomarker analytes. As a proof of concept, we
quantify human epididymis protein 4 (HE4) at low pM levels in a few
hours. Future improvement of this method by integration with a nanopore
array and microfluidics-based chemistry can further reduce the limit
of detection, allow multiplexed biomarker detection, and further reduce
the footprint and cost of existing laboratory and point-of-care devices.
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