Programmable Nucleic Acid Based Polygons with Controlled Neuroimmunomodulatory Properties for Predictive QSAR Modeling

Abstract

In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

Keywords: QSAR; RNA and DNA nanoparticles; RNA nanotechnology; RNA polygons; immunology; self-assembly.

Figure 1:

Programmable nucleic acid RNA (A) and DNA (B) polygons. Each panel presents energy minimized 3D models of RNA and DNA nanoparticles (identical sequences are colored the same), with corresponding AFM images, hydrodynamic radii measured by DLS (presented as +/− SEM), and ethidium bromide total staining native-PAGE results. “MW standards” denote the low molecular weight DNA ladder (NEB) used as the size marker.

Figure 2:

Cell culture experiments with programmable polygons. Human microglia-like cell lines were transfected with polygons at a final concentration of 5 nM and 25 nM. (A) 3D models of tested polygons with RNA strands shown in grey and DNA strands in blue. (B) Assemblies all polygons visualized by agarose gel. “MW st.” denotes the low molecular weight DNA ladder (NEB). (C) Structural integrity of polygons associated with Lipofectamine 2000 (L2K) confirmed by the release studies with Triton X100. The results are analyzed by native-PAGE and visualized by AFM. (D) Relative cellular uptakes assessed by flow cytometry and (E) cell viability assays of polygons transfected with L2K. Polygons tested without L2K showed negligible change in cell viability (data not shown). (F-G) 24 hours post transfection with 16 RNA, DNA and RNA/DNA polygons, cell supernatants were collected and levels of IFN-β (F) and IL-6 (G) production were assessed by specific-capture ELISA. In C, E, and F, results were normalized to transfection reagent alone treated cells (L2K) and presented as the mean +/− SEM. Statistically significant results are indicated with asterisks (p value < 0.05).

Figure 3:

Schematic representation of quantitative structure-activity relationship (QSAR) modeling used in this project.