Dynamic Behavior of RNA Nanoparticles Analyzed by AFM on a Mica/Air Interface

Abstract

RNA is an attractive biopolymer for engineering self-assembling materials suitable for biomedical applications. Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduce gaps into the double-stranded regions of the RNA rings. Molecular dynamic simulations are used to assess the dynamic behavior and the changes in the flexibility of novel designs. The changes suggested by simulations, however, cannot be clearly confirmed by the conventional techniques such as nondenaturing polyacrylamide gel electrophoresis (native-PAGE) and dynamic light scattering (DLS). Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of new assemblies. To address these deficiencies, we introduce a computer-assisted quantification strategy. This strategy is based on an algorithmic atomic force microscopy (AFM)-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validate this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis are anticipated to provide a broad spectrum approach for the future development of nucleic acid-based nanotechnology.

Toll-Like Receptor-Mediated Recognition of Nucleic Acid Nanoparticles (NANPs) in Human Primary Blood Cells

“…Based on our earlier findings [21,22,23,33,42,43] four of the most representative NANPs were chosen as a model system for this study. We chose a small representative group of NANPs to address the influence of their shape (RNA cubes vs. planar RNA rings vs. RNA fibers), and composition (RNA cube vs. DNA cube).…”

“…For example, NANP technologies use general knowledge of the structures and biological functions of various natural and artificial classes of RNAs (or DNAs) to tackle specific biochemical problems. These novel assemblies have been extensively characterized in vitro (e.g., by electrophoretic mobility shift assay (EMSA) [18], cryogenic electron microscopy (cryo-EM) [19,20], atomic force microscopy (AFM) [21,22,23], and dynamic light scattering (DLS) [23,24] and demonstrated to effectively operate in vivo [20,25,26,27,28,29,30,31,32]. However, the immunorecognition of novel NANPs is widely unknown and can preclude their further biomedical applications.…”

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles

“…Likewise, RNA squares were less immunostimulatory than RNA cubes containing 6 ssUs per 3WJ even though both materials had a total of 48 ssUs per NANP (Figure 3C). Interestingly, introduction of the ssNTs’ regions into RNA rings 52 did not make any difference in IFN induction by these particles (Figure S10). Collectively, these data suggest that the immunostimulatory effect of ssNTs is unique to the 3D RNA NANPs (i.e., RNA cubes).…”

Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology

“…Tracing changes in FRET signals that result upon re-hybridization of labeled strands entering the compositions of cognate NANPs confirms the dynamicity of their behavior in real time [18,21,22]. The integrity of NANPs in cells can be verified through the co-localization of multiple different fluorophores simultaneously entering the composition of NANPs [23,24]. RNA probes also allow for a modular approach to visualization; fluorescent in situ hybridization, or FISH, and molecular beacons have been introduced to cells to give a fluorescent response upon binding to a target sequence [25,26,27].…”