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Defining the Structure of a Protein-Spherical Nucleic Acid Conjugate and Its Counterionic Cloud

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Journal Article - Open Access

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Northwestern University Evanston United States

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Proteinspherical nucleic acid conjugates Pro-SNAs are an emerging class of bioconjugates that have properties defined by their protein cores and dense shell of oligonucleotides. They have been used as building blocks in DNA-driven crystal engineering strategies and show promise as agents that can cross cell membranes and affect both protein and DNA-mediated processes inside cells. However, ionic environments surrounding proteins can influence their activity and conformational stability, and functionalizing proteins with DNA substantively changes the surrounding ionic environment in a nonuniform manner. Techniques typically used to determine protein structure fail to capture such irregular ionic distributions. Here, we determine the counterion radial distribution profile surrounding Pro-SNAs dispersed in RbCl with 1 nm resolution through in situ anomalous small-angle X-ray scattering ASAXS and classical density functional theory DFT. SAXS analysis also reveals the radial extension of the DNA and the linker used to covalently attach the DNA to the protein surface. At the experimental salt concentration of 50 mM RbCl, Rb cations compensate 90 of the negative charge due to the DNA and linker. Above 75 mM, DFT calculations predict overcompensation of the DNA charge by Rb. This study suggests a method for exploring Pro-SNA structure and function indifferent environments through predictions of ionic cloud densities as a function of salt concentration, DNA grafting density, and length. Overall, our study demonstrates that solution X-ray scattering combined with DFT can discern counterionic distribution and submolecular features of highly charged, complex nanoparticle constructs such as Pro-SNAs and related nucleic acid conjugate materials.

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