Aggregation and Oligomerization Characterization of ß-Lactoglobulin Protein Using a Solid-State Nanopore Sensor.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (ßLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing ßLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the ßLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, ßLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore.

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.

Estimating RNA Polymerase Protein Binding Sites on λ DNA Using Solid-State Nanopores.

In this work, using a silicon nitride nanopore based device, we measure the binding locations of RNA Polymerase (RNAP) on 48.5 kbp (16.5 µm) long λ DNA. To prevent the separation of bound RNAPs from a λ DNA molecule in the high electric field inside a nanopore, we cross-linked RNAP proteins to λ DNA by formaldehyde. We compare the current blockage event data measured with a mixture of λ DNA and RNAP under cross-link conditions with our control samples: RNAP, λ DNA, RNAP, and λ DNA incubated in formaldehyde separately and in a mixture. By analyzing the time durations and amplitudes of current blockage signals of events and their subevents, as well as subevent starting times, we can estimate the binding efficiency and locations of RNAPs on a λ DNA. Our data analysis shows that under the conditions of our experiment with the ratio of 6 to 1 for RNAP to λ DNA molecules, the probability of an RNAP molecule to bind a λ DNA is ∼42%, and that RNAP binding has a main peak at 3.51 µm ± 0.53 µm, most likely corresponding to the two strong promoter regions at 3.48 and 4.43 µm of λ DNA. However, individual RNAP binding sites were not distinguished with this nanopore setup. This work brings out new perspectives and complications to study transcription factor RNAP binding at various positions on very long DNA molecules.