Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10–25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.

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Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

Recent advances in instrumentation have moved analytical ultracentrifugation (AUC) closer to a possible validation in a Good Manufacturing Practices (GMP) environment. In order for AUC to be validated for a GMP environment, stringent requirements need to be satisfied; analysis procedures must be evaluated for consistency and reproducibility, and GMP capable data acquisition software needs to be developed and validated. These requirements extend to multiple regulatory aspects, covering documentation of instrument hardware functionality, data handling and software for data acquisition and data analysis, process control, audit trails and automation. Here we review the requirements for GMP validation of data acquisition software and illustrate software solutions based on UltraScan that address these requirements as far as they relate to the operation and data handling in conjunction with the latest analytical ultracentrifuge, the Optima AUC by Beckman Coulter. The software targets the needs of regulatory agencies, where AUC plays a critical role in the solution-based characterization of biopolymers and macromolecular assemblies. Biopharmaceutical and regulatory agencies rely heavily on this technique for characterizations of pharmaceutical formulations, biosimilars, injectables, nanoparticles, and other soluble therapeutics. Because of its resolving power, AUC is a favorite application, despite the current lack of GMP validation. We believe that recent advances in standards, hardware, and software presented in this work manage to bridge this gap and allow AUC to be routinely used in a GMP environment. AUC has great potential to provide more detailed information, at higher resolution, and with greater confidence than other analytical techniques, and our software satisfies an urgent need for AUC operation in the GMP environment. The software, including documentation, are publicly available for free download from Github. The multi-platform software is licensed by the LGPL v.3 open source license and supports Windows, Mac and Linux platforms. Installation instructions and a mailing list are available from ultrascan.aucsolutions.com.

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135–555 interacting with and unwinding RVFV non-coding regions.