One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize or unfold β-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration.

One primary goal in nanobiotechnology is designing new methodologies for molecular biomedical diagnosis at stages much earlier than currently possible and without use of expensive reagents and sophisticated equipment. In this work, we show the proof of principle for single-molecule detection of the nucleocapsid protein 7 (NCp7), a protein biomarker of the HIV-1 virus, using synthetic nanopores and the resistive-pulse technique. The biosensing mechanism relied upon specific interactions between NCp7 and aptamers of stem-loop 3 (SL3) in the packaging domain of the retroviral RNA genome. One critical step of this study was the choice of the optimal size of the nanopores for accurate, label-free determinations of the dissociation constant of the NCp7 protein - SL3 RNA aptamer complex. Therefore, we systematically investigated the NCp7 protein - SL3 RNA aptamer complex employing two categories of nanopores in a silicon nitride membrane: (i) small, whose internal diameter was smaller than 6 nm, and (ii) large, whose internal diameter was in the range of 7 through 15 nm. Here, we demonstrate that only the use of nanopores with an internal diameter that is smaller than or comparable with the largest cross-sectional size of the NCp7-SL3 aptamer complex enables accurate measurement of the dissociation constant between the two interacting partners. Notably, this determination can be accomplished without the need for prior nanopore functionalization. Moreover, using small solid-state nanopores, we demonstrate the ability to detect drug candidates that inhibit the binding interactions between NCp7 and SL3 RNA by using a test case of N-ethylmaleimide.

5-bisphosphate carboxylase/oxygenase (RuBisCO). The challenge associated with structure and functional investigation of Rca can be attributed to its exceptionally low thermo-stability, high degree of size polydispersity and tendency toward subunit aggregation. In this work we have successfully employed fluorescence fluctuation methods to study the nucleotide-dependent stoichiometry of fluorescently tagged Rca for a wide range of concentrations. Our results show a stepwise assembly pathway of Rca under different assay conditions. In presence of Mgþ2 -ADP, the oligomerization state of Rca is largely dominated by monomers at concentrations below 0.5 mM. The state of oligomerization gradually changes in steps of two subunits. The most probable model for this assembly supports the dissociation coefficients of~4, 1, 1 mM for the monomer-dimer, dimer-tetramer and tetramer-hexamer equlibria respectively. Continued assembly at even higher concentrations suggests self association through the formation of spiral arrangements that grow along the helical axis. 2706-Pos Board B725Inferring Subunit Stoichiometry from Single Molecule Photobleaching Keegan E. Hines. University of Texas at Austin, Austin, TX, USA. Single molecule photobleaching is a powerful tool for determining the stoichiometry of protein complexes. By attaching fluorophores to proteins of interest, the number of associated subunits in a complex can be deduced by imaging single molecules and counting fluorophore photobleaching steps. Since some bleaching steps might be unobserved, the ensemble of bleaching steps will be binomially distributed and it has been commonly assumed that the highest number of observed bleaching steps is indicative of the stoichiometry of the complex. However, we point out that inferring the true composition of a complex from such data is non-trivial because binomial processes are ill-posed. As a result, there may be a significant probability that the true complex is larger than the data indicate simply due to finite sample size and the variance of binomial processes. Because of this possibility, calculating likelihoods to establish parameter confidence can be misleading. What is needed is a reliable method to quantify one's conclusions about stoichiometry. We present a Monte Carlo method which does not rely on likelihood calculation and provides a reliable estimate of confidence. The formalization and methods presented here provide a rigorous analytical basis to this pervasive experimental tool. Nanopores are fast becoming a major scientific tool in molecular analysis and detection due to their ability to detect polynucleotides, proteins, and small molecules. Previous work has included the manipulation of a-hemolysin into a DNA sequencing pore however further development is needed for the use of a simple, monomeric pore for this purpose. Biomimetic modelling of nanopores allows for a specific function to be built into the model based on the replication and analysis of existing selectivity present in proteins. An initial analysis of know...

In the Results and Discussions section of our article, the last term in Equation (3) should read 1/d not 1/d^2. The correct form of the full equation is, This erratum does not affect any of the experimental results, discussions, or conclusions reported in the paper. The authors sincerely apologize for this unintended oversight.

Background: Tau is a neuronal scaffolding protein which aggregates intracellularly upon hyperphosphorylation to form neurofibrillary tangles (NFT). NFTs are a hallmark of various microtubule associated protein tau (MAPT)‐associated neurodegenerative diseases, including Alzheimer’s disease, frontotemporal dementia, and progressive supranuclear palsy. Currently, there is no approved treatment targeting Tau pathology, nor any other disease‐modifying therapy for these diseases. Wave Life Sciences has developed a stereopure antisense oligonucleotide to reduce MAPT expression in patients with MAPT‐associated neurodegenerative diseases. Method: Using Wave’s proprietary PRISM platform, we designed and screened stereopure oligonucleotides targeting MAPT in vitro and identified a lead sequence. PN chemistry was applied to the lead sequence in subsequent studies to further improve pharmacological properties. In vitro target engagement was measured in human iCell neurons after treatment with Wave’s oligonucleotide, WVE‐005, or a published reference stereorandom oligonucleotide under gymnotic conditions. For in vivo analysis, transgenic mice received a single dose of 12.5, 25, 50, or 100µg oligonucleotide, and target engagement was measured after 4 weeks. To measure duration, transgenic mice received a single 100µg ICV dose and target engagement was tracked over 6 months. Finally, oligonucleotides were tested in non‐human primates (NHP) in a 12mg single‐dose intrathecal (IT) study. MAPT mRNA levels were quantified by qPCR, and intracellular distribution was evaluated by ViewRNA. Result: WVE‐005 showed dose‐dependent silencing of MAPT mRNA in iPSC‐derived neurons with an IC50 of 84nM. In a dose‐response study, 12.5µg and 25µg WVE‐005 led to 50% knockdown after 4 weeks in hippocampus and cortex, respectively, in transgenic mice. WVE‐005 led to >77% MAPT mRNA knockdown at 12 weeks post 100µg dosing, with knockdown of approximately 50% persisting 6 months post‐injection. In NHPs, WVE‐005 showed improved distribution and potency compared to non‐PN oligonucleotide with the same sequence. WVE‐005 decreased MAPT expression across NHP brain regions 28 days post‐single dose much greater than the non‐PN oligonucleotide and was detected in neuronal and glial cells. Conclusion: WVE‐005 potently and durably decreased MAPT mRNA expression in vitro and in multiple animal models, including throughout CNS upon IT administration in NHPs. These data support continued evaluation of WVE‐005 as a potential therapeutic for MAPT‐associated neurodegenerative diseases.