Multiwavelength Analytical Ultracentrifugation (MW-AUC)

Multiwavelength analytical ultracentrifugation (MW-AUC) is an advanced technique that extends the hydrodynamic information collected by analytical ultracentrifugation (AUC). Analytical ultracentrifugation (AUC) measures partial concentrations, sedimentation coefficients, and diffusion coefficients of analytes in colloidal mixtures, providing insights into their size and anisotropy, typically at a single wavelength, in the ultraviolet or visible range.

MW-AUC collects data from multiple wavelengths during an experiment, enabling characterization by resolving analytes based on their hydrodynamic properties and absorbance spectra. By collecting data at multiple wavelengths, MW-AUC can distinguish between different species in a mixture based on their distinct absorbance spectra. This makes it possible to distinguish between various components in complex mixtures, a feat that was challenging using traditional methods. MW-AUC can provide more accurate quantification of the concentration and distribution of different components in a sample.¹ This is because it reduces the reliance on a single wavelength, which might not be optimal for all components present.

Combining the multiwavelength detector system and advanced analysis tools enables researchers to explore and understand complex molecular interactions and precisely characterize each component. MW-AUC has enhanced the analysis of many interacting systems, including protein-DNA,^2,3 protein-RNA,⁴ biopolymers⁵ heme proteins and amyloid-β peptide interactions,⁶ as well as the characterization of AAVs.^1,7

a)

b)

c)

Figure 1: Multiwavelength AUC results of Human Serum Albumin (HAS) mixed with porphyrin. The multiwavelength experiment is seen in (a) plotting the hydrodynamic properties as a function of wavelength depicting spectral properties. (b) is an example of a single wavelength study at 278 nm, where two species are observed but cannot be discerned for each other. (c) is the results of the multiwavelength AUC analysis, from which baseline separation of the two peaks is achieved with apo-HAS shown in red and HSA complexed with porphyrin. To read this study, visit this page.

Applications of Multiwavelength Analytical Ultracentrifugation (MW-AUC)

MW-AUC provides a robust platform for advanced biopolymer analysis, leveraging hydrodynamic and spectral data to achieve precise characterization. Key applications include:

1. INTERACTION/BINDING STUDIES HYDRODYNAMIC SEPARATION OF SPECTRAL COMPONENTS
   
   • AUC separates and resolves biopolymers in solution based on unique hydrodynamic properties.
   • Multiwavelength adds the spectral separation of biopolymers on top of the hydrodynamic separation, allowing for accurate quantification and determination of molar ratios of binding partners.
   • Utilizes additional spectral data to distinguish analytes by their unique chromophores, providing orthogonal characterization.
2. DRUG-LOADED LIPID NANOPARTICLE (LNP) IDENTIFICATION^8,9
   
   • Identifies and characterizes drug-loaded LNPs through spectral markers:
     • 260 nm: Absorption peak for nucleic acids.
     • 490 nm: Prominent absorption peak for Doxyrubicin (a drug).
     • 230 nm: High scattering signal for liposomes and lipid nanoparticles.
3. DIFFERENTIATION OF AAV CAPSIDS
   
   • Distinguishes between empty, full and partially filled AAV capsids using wavelength-specific absorption data:
     • 230 nm: Capsid protein peptide bonds and Mie scattering.
     • 260 nm: DNA absorption.
     • 280 nm: Capsid protein aromatic sidechain absorption.

MW-AUC's dual-dimensional data acquisition significantly enhances the resolution and specificity of analyte identification and characterization, making it indispensable in advanced molecular research⁶.

Advantages of MW-AUC

MW-AUC offers several distinct advantages, making it a valuable tool for advanced molecular and biophysical studies:

1. ENHANCED ANALYTICAL CAPABILITIES
   
   • Adds a spectral characterization dimension to traditional hydrodynamic separation achieved by analytical ultracentrifugation (AUC).
   • Identifies and quantifies analytes in solution based on their unique spectral properties.
   • Distinguishes components in mixtures with overlapping absorbance at a single wavelength.
   • More precise quantification of each component in solution.
2. VERSATILITY
   
   • Adapts to various sample types, including proteins, nucleic acids, polymers, nanoparticles and complex mixtures.
3. NON-DESTRUCTIVE METHODOLOGY
   
   • Preserves sample integrity, allowing for further downstream analyses.
   • Few buffer constraints.
   • No dye required.
4. BINDING STUDIES
   
   • Facilitates detailed investigation of molecular interactions, including:
     • Protein-protein binding.
     • Protein-DNA interactions.
     • Protein-ligand binding.
   • Differentiates bound and unbound states through their distinct spectral signatures.

Multiwavelength Protocol

MW-AUC measures samples at multiple wavelengths, typically between 15 and 28, although experiments with only 3 wavelengths can also be beneficial and are referred to as multiwavelength. If a larger number of wavelengths is selected (>15),^1,6 optical deconvolution of the data can be performed. It is important to select wavelengths where the spectra of the macromolecules in solution differ from each other, aiming to maximize the spectral angle between spectral partners.

After collecting all the data, a 3D plot can be created to show the spectral signal against the sedimentation coefficient. This helps visualize the sample and highlights where different components in the solution sediment.⁶ If the spectral properties of the components are known, the MW data can be separated into their spectral contributors. The MW dataset is then decomposed into traditional datasets for further analysis, revealing the behavior and interactions of different spectral analytes. This dataset can also be decomposed into their molar concentrations if the extinction coefficients are known, allowing for the determination of the molar ratio of binding partners.

References

1. Maruno T, Usami K, Ishii K, Torisu T, Uchiyama S. Comprehensive Size Distribution and Composition Analysis of Adeno-Associated Virus Vector by Multiwavelength Sedimentation Velocity Analytical Ultracentrifugation. J Pharm Sci. 2021 Oct;110(10):3375–3384. PMID: 34186069
2.  Ahmed I, Hahn J, Henrickson A, Khaja FT, Demeler B, Dubnau D, Neiditch MB. Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria. Nat Commun. 2022 Dec 13;13(1):7724. PMCID: PMC9747964
3. Horne CR, Venugopal H, Panjikar S, Wood DM, Henrickson A, Brookes E, North RA, Murphy JM, Friemann R, Griffin MDW, Ramm G, Demeler B, Dobson RCJ. Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism. Nat Commun. 2021 Mar 31;12(1):1988. PMCID: PMC8012715
4. Zhang J, Pearson JZ, Gorbet GE, Cölfen H, Germann MW, Brinton MA, Demeler B. Spectral and Hydrodynamic Analysis of West Nile Virus RNA-Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge. Anal Chem. 2017 Jan 3;89(1):862–870. PMCID: PMC5505516
5. Johnson CN, Gorbet GE, Ramsower H, Urquidi J, Brancaleon L, Demeler B. Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin. Eur Biophys J. 2018 Oct;47(7):789–797. PMCID: PMC6158097
6. Henrickson A, Gorbet GE, Savelyev A, Kim M, Hargreaves J, Schultz SK, Kothe U, Demeler B. Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions. Anal Biochem. 2022 Sep 1;652:114728. PMCID: PMC10276540
7. Henrickson A, Ding X, Seal AG, Qu Z, Tomlinson L, Forsey J, Gradinaru V, Oka K, Demeler B. Characterization and quantification of adeno-associated virus capsid-loading states by multi-wavelength analytical ultracentrifugation with UltraScan. Nanomedicine (Lond). 2023 Sep;18(22):1519–1534. PMCID: PMC10652292
8. Henrickson A, Kulkarni JA, Zaifman J, Gorbet GE, Cullis PR, Demeler B. Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations. ACS Nano. 2021 Mar 23;15(3):5068–5076. PMID: 33617224
9. Bhattacharya A. How does data from AUC determine dose optimization in therapeutic liposomes? [Internet]. [cited 2024 Dec 27]. Available from: https://www.beckman.com/resources/techniques-and-methods/spinsights/issue-8

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