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.

Real-time shape approximation and fingerprinting of single proteins using a nanopore.

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

The effects of geometry and stability of solid-state nanopores on detecting single DNA molecules.

In this work we use a combination of 3D-TEM tomography, energy filtered TEM, single molecule DNA translocation experiments, and numerical modeling to show a more precise relationship between nanopore shape and ionic conductance and show that changes in geometry while in solution can account for most deviations between predicted and measured conductance. We compare the structural stability of ion beam sculpted (IBS), IBS-annealed, and TEM drilled nanopores. We demonstrate that annealing can significantly improve the stability of IBS made pores. Furthermore, the methods developed in this work can be used to predict pore conductance and current drop amplitudes of DNA translocation events for a wide variety of pore geometries. We discuss that chemical dissolution is one mechanism of the geometry change for SiNx nanopores and show that small modification in fabrication procedure can significantly increase the stability of IBS nanopores.