Spectral and Hydrodynamic Analysis of West Nile Virus RNA–Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge

Abstract

Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein—RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable.

LED based near infrared spectral acquisition for multiwavelength analytical ultracentrifugation: A case study with gold nanoparticles

“…For the analysis of MWL broad spectral bandwidth samples involving fitting for sedimentation and diffusion coefficient distributions, and the application of computationally intensive operations such as Monte-Carlo algorithms, the availability of supercomputer methods in programs such as Ultra-Scan3 are very powerful. The two-dimensional spectral analysis (2DSA) and parametrically constrained spectral analysis (PCSA) employed in UltraScan3 have proven especially useful [15,41,42].…”

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

“…This is easily achieved when the intrinsic extinction profile for the interacting partners is sufficiently different, for example, when comparing protein and DNA spectra 53 . Once deconvoluted and on a molar scale, the stoichiometry of the complex can simply be extracted by integrating the molar ratio of the co-migrating peaks 55 . Thus, multiwavelength sedimentation velocity experiments provide both hydrodynamic and spectral characterization of an interacting system to define the stoichiometry of association, as well as the hydrodynamic properties such as the mass and frictional ratio of each species.…”

“…Molar extinction profiles were determined by performing a dilution series for both NanR and DNA by collecting an absorbance spectrum across the spectral range of interest (220-300 nm) using a Genesys 10s benchtop spectrophotometer (Thermo Fisher Scientific). The dilution series of each absorbance spectra was fitted to intrinsic extinction profiles as described previously 53,55 . The resulting intrinsic extinction profiles were scaled to molar concentration using an extinction coefficient of 13,980 M -1 cm -1 at 280 nm for wild-type NanR and for NanR as calculated by ExPASy ProtParam from the amino acid sequence.…”

“…An angle of 0° reflects linear dependence or perfect overlap, while an angle of 90° indicates perfect orthogonality or no spectral overlap. Next, the spectral profiles scaled to molar concentration were subsequently used to deconvolute the noise-corrected multiwavelength data into separate datasets for the NanR and DNA components using the non-negatively constrained least squares algorithm 54 as previously described 53,55 . These deconvoluted datasets were individually analyzed by the 2DSA method using UltraScan 31 .…”

Practical Aspects of Multiwavelength Analytical Ultracentrifugation

“…This manuscript provides additional details that have not yet been described or were more recently adapted, but have proven essential to producing high quality measurements from the instrument. We hope to present a realistic portrayal of the challenges a researcher faces when considering implementing one of these systems in their own laboratory, in addition to many of the critical aspects of the installation that we have found necessary for successful instrument operation demonstrated in past publications [2,[6][7][8]. This includes a description of the necessary tools and expertise to build and install the detector, followed by a protocol for alignment and calibration.…”