Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Abstract

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals. STATEMENT OF SIGNIFICANCENanopore sensing allows investigation of biomolecular structure in aqueous solution, including electricfield-induced changes in protein conformation. This nanopore-based study probes the tetramer-dimer transition of streptavidin, observing the effects of increasing voltage with varying salt type and concentration. Binding of biotinylated DNA to streptavidin boosts the complex's structural integrity, while complicating signal analysis. We describe a broadly applicable geometric approach that maps stepwise changes in the nanopore signal to real-time conformational transitions. These results represent important progress toward accurate interpretation of nanopore signals generated by macromolecular complexes.

Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons

“…The degree with which the nanopore sensor can report the detection of these molecules is a function of the pore properties (pore size, surface chemistry), operating condition (salt concentration, pH, voltage), the properties of the off-target molecules in the sample (size, charge, and propensity to stick to and clog pores in a given set of operating conditions), and the response time of the amplifier-pore system. [20][21][22][23][24][25][26][27][28] Consequently, we first performed control experiments to investigate the nanopore response to different PCR mixtures to identify adequate operating conditions for sensing amplicons.…”

Modulation of electrophoresis, electroosmosis and diffusion for electrical transport of proteins through a solid-state nanopore

“…While high bandwidth equipment would permit to detect fast moving protein events, as noted by Tabard Cossa et al, we obtained statistically signicant data (>1000 events) across all events to draw conclusions based on events slowed down through interactions with the pore. 44 At higher electric elds, experiments with 0.5 M LiCl were not possible for two reasons: persistent clogging and poor C R at high electric elds. Moreover, as mentioned, hSTf continues to unfold beyond 400 mV in 4 M LiCl and 1 M LiCl while DG p,f plateaus at $400 mV in 2 M KCl.…”

“…However, we note that the bandwidth limitations of the Axopatch 200B amplifier may cause ballistic events to be not detected and only those that are delayed through interactions with the pore to be detected as noted by previous work in the literature. 44 To affirm the missed events, we first calculated the effective capture radius ( r eff ) using r eff = C R /2 πDC where D and C are bulk diffusion coefficient (estimated using the Stokes–Einstein equation and was found to be ∼7.6 × 10 −7 cm 2 s −1 for hSTf) and hSTf concentration (∼100 nM) respectively. The r eff at ±400 mV was found to be ∼1 nm, ∼0.6 nm and ∼0.9 nm in 2 M KCl, 4 M LiCl and 1 M LiCl respectively which is at least 14× smaller than the pore radius.…”

“…While high bandwidth equipment would permit to detect fast moving protein events, as noted by Tabard Cossa et al , we obtained statistically significant data (>1000 events) across all events to draw conclusions based on events slowed down through interactions with the pore. 44 …”

Highlights on the current state of proteomic detection and characterization with nanopore sensors

“…Carlson and Tabard‐Cossa [19] perform a thorough analysis of resistive pulses resulting from proteins and protein–DNA complexes moving through a solid‐state nanopore. Their goal is to better understand the origins of the blockade events under different voltages, salts, and ionic strengths in the hopes of improving the sensing capabilities of solid‐state pores.…”