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

Abstract

Protein–spherical 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 in different 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.

Programming colloidal bonding using DNA strand-displacement circuitry

“…Programming PAE Bonding for the Formation of Binary AuNP-Protein Lattice. The programmable coassembly of multiple nanoscale building blocks with disparate chemical and physical properties, including different inorganic nanoparticles (e.g., Fe 3 O 4 , Pt, and quantum dots) (5, 13) and biomolecules (e.g., proteins) (35)(36)(37) is of great potential in the applications of catalysis, sensing, and photonics. However, for thermal annealing, the strict melting temperature limits the participation of biomolecules, because the intrinsic temperature-sensitive biomolecule such as protein may suffer from possible denaturation upon annealing treatment at a high temperature.…”

Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery

“…Moreover, neither the spherical shape or core materials is an important factor to govern the endocytosis. Advanced endocytic uptake has been reported on the SNA that siRNA are assembled on gold nanorod, liposome and globular protein [129,130,131].…”

ASAXS measurements on ferritin and apoferritin at the bioSAXS beamline P12 (PETRA III, DESY)

“…ASAXS has been successfully applied to investigate the ion distributions around polyelectrolytes, DNA and micelles (Ballauff & Jusufi, 2006;Pabit et al, 2010;Dingenouts et al, 2004;Jusufi et al, 2004). The combination of ASAXS with density functional theory has allowed deeper insights and validation of structural models on the counter-ion distribution and sub-molecular features (Krishnamoorthy et al, 2018). Conventional smallangle X-ray scattering (SAXS) measurements cannot easily deduce such information as the scattering intensities of the macromolecules, and the associated ions are convoluted into the scattering amplitudes proportionally to their volume and contrast.…”