The dynamic, motile and deformative properties of RNA nanoparticles facilitate the third milestone of drug development

Abstract

Besides mRNA, rRNA, and tRNA, cells contain many other noncoding RNA that display critical roles in the regulation of cellular functions. Human genome sequencing revealed that the majority of non-protein-coding DNA actually codes for non-coding RNAs. The dynamic nature of RNA results in its motile and deformative behavior. These conformational transitions such as the change of base-pairing, breathing within complemented strands, and pseudoknot formation at the 2D level as well as the induced-fit and conformational capture at the 3D level are important for their biological functions including regulation, translation, and catalysis. The dynamic, motile and catalytic activity has led to a belief that RNA is the origin of life. We have recently reported that the deformative property of RNA nanoparticles enhances their penetration through the leaky blood vessel of cancers which leads to highly efficient tumor accumulation. This special deformative property also enables RNA nanoparticles to pass the glomerulus, overcoming the filtration size limit, resulting in fast renal excretion and rapid body clearance, thus low or no toxicity. The biodistribution of RNA nanoparticles can be further improved by the incorporation of ligands for cancer targeting. In addition to the favorable biodistribution profiles, RNA nanoparticles possess other properties including self-assembly, negative charge, programmability, and multivalency; making it a great material for pharmaceutical applications. The intrinsic negative charge of RNA nanoparticles decreases the toxicity of drugs by preventing nonspecific binding to the negative charged cell membrane and enhancing the solubility of hydrophobic drugs. The polyvalent property of RNA nanoparticles allows the multi-functionalization which can apply to overcome drug resistance. This review focuses on the summary of these unique properties of RNA nanoparticles, which describes the mechanism of RNA dynamic, motile and deformative properties, and elucidates and prepares to welcome the RNA therapeutics as the third milestone in pharmaceutical drug development.

Keywords: Cancer treatment; Deformative property; Drug delivery; Drug development; RNA dynamics; RNA nanotechnology; Ribonucleic acid (RNA).

Fig. 1.

RNA hierarchical structure and dynamics. (A) Secondary and (B) tertiary structures of tRNA (Lys, 3). Reprinted with permission from [51]. Copyright 2010 Molcular Diversity Preservation International; (C) Quaternary structure of phi29 pRNA hexamer. Adapted with permission from [57]. Copyright 2021 American Chemical Society; (D) Three tiers of RNA dynamics in hierarchical free-energy landscape. Adapted with permission from [14]. Copyright 2019 Springer Nature Limited.

Fig. 2.

Melting temperature and nearest neighbor dynamics of RNA nanoparticles. (A) TGGE gels and their quantification curves of 3WJ-10 PTXs and 4WJ-X-24 PTXs RNA nanoparticles. Reprinted with permission from [28]. Copyright 2020 The Author(s); (B) Secondary structure of pRNA with central 3WJ motif (red circle) composing a_3WJ, b_3WJ, and c_3WJ strands. Reprinted with permission from [78]. Copyright 2013 Oxford University Press; (C) The strategy of constructing RNA complexes harboring multiple functional groups (1–4) driven by the formation of 3WJ core. Reprinted with permission from [78]. Copyright 2013 Oxford University Press.

Fig. 3.

RNA dynamics at structure and function level. (A) Folding topology and 3D structural model of the phage T2 gene 32 autoregulatory pseudoknot (PBD 2TPK). Adapted with permission from [79]. Copyright 2008 Elsevier B.V.; (B) DNA breathing demonstrated by dsDNA with the breaking A-T base pair (green) generated from atomistic MD simulation. Reprinted with permission from [84]. Copyright 2016 Oxford University Press; (C) Mechanism of FMN recognition by induced fit under crowding conditions with PEG200 and by conformational capture under dilute condition with MgCl₂. Reprinted with permission from [89]. Copyright 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinhem.

Fig. 4.

RNA dynamics before, during, and after intrinsic equilibrium. (A) Pre-rearranged structure and post-rearranged structure with the formation of extended helix (yellow) of E. coli SPR RNA during cotranscriptional folding. Reprinted with permission from [90]. Copyright 2020 Elsevier Inc.; (B) Three conformation of TAR with interhelical motions (Black: Helix I; Orange: Helix II). Reprinted with permission from [62]. Copyright 2007 Nature Publishing Group; (C) The transition of riboswitch from on to off status after ligand (yellow) binding. Reprinted with permission from [16]. Copyright 2008 Elsevier Ltd.

Fig. 5.

Programmability of RNA nanoparticles over (A) shape; (B) size; (C) stability; and (D) stoichiometry. (A) Schematic and AFM image of triangle, square, and pentagon RNA nanoparticles. Adapted with permission from [97]. Copyright 2014 Oxford University Press; (B) Schematic, size measurement, and AFM image of 5, 10, and 20 nm square RNA nanoparticles. Adapted with permission from [21]. Copyright 2014 American Chemical Society; (C) Secondary structure and melting profile of three 3WJs with different sequences. Adapted with permission from [105]. Copyright 2020 Royal Society of Chemistry; (D) Self-assembly of RNA dendrimers with 18 PTXs (yellow) and 3 FAs (magenta) using RNA strands with 6 PTXs, with 1 FA, and without modification. Reprinted with permission from [105]. Copyright 2020 Royal Society of Chemistry.

Fig. 6.

Deformative ability of RNA nanoparticles contributes to favorable biodistribution. (A) Biodistribution comparison of iron, gold, and RNA nanoparticles with IVIS images of tumors and organs, and the fluorescent quantification of tumor to organ (liver and kidney) ratio; (B) Gel image of urine samples at 0.5 hr post IV injection of dsRNA, 3WJ, and 4WJ RNA nanoparticles; (C) IVIS images of organs and tumors collected at 12 and 24 hr post IV injection of 10 nm and 20 nm RNA square nanoparticles. Adapted with permission from [25]. Copyright 2020 American Chemical Society.

Fig. 7.

Active targeting of RNA nanoparticles with small molecule ligand (A, B, C) and RNA aptamer (D, E, F). (A) Design of Phi29 3WJ with Alexa₆₄₇ fluorescent dye and FA targeting ligand; (B) Fluorescent confocal images of liver metastasis and adjacent healthy liver at 6 hr post IV injection of PBS and RNA nanoparticles. (Blue: DAPI; Green: GFP expressing cancer cells; Red: Alexa₆₄₇); (C) Alexa (top panel) and GFP (bottom panel) fluorescent images of liver with metastasis at 6 hr post IV injection of PBS, pRNA and FA-pRNA. Adapted with permission from [112]. Copyright 2015 American Chemical Society; (D) Design of Phi29 3WJ with Alexa₆₄₇ fluorescent dye and PSMA RNA aptamer; (E) Fluorescent confocal images of tumor samples at 8 hr post IV injection of RNA nanoparticles (Blue: DAPI; Red: Alexa₆₄₇); (F) Biodistribution of A9g-3WJ and 3WJ in organs and tumors demonstrated by Alexa₆₄₇ signal. Adapted with permission from [38]. Copyright 2016 American Society of Gene & Cell Therapy.

Fig. 8.

RNA nanoparticles improve water solubility of drug and overcome drug resistance. RNA increases water solubility of CPT (A) and PTX (B) Adapted with permission from [28,29]. Copyright 2019 The Authors and 2020 The Author(s); (C) Design of multifunctional 3WJ RNA nanoparticle with MED siRNA and HER2 aptamer; (D) IVIS lumina images of tumors in mice bearing breast tumor after treatment. Adapted with permission from [30]. Copyright 2016 American Chemical Society; (E) Design of multifunctional 6WJ RNA nanoparticle with PTX, miR122, and HTL; (F) Synergistic cytotoxic effect of PTX and miR122 using MTT assay and HSA synergy model; (G) Image and quantification of tumors harvest from mice bearing HCC tumor after treatment. Adapted with permission from [31]. Copyright 2020 Elsevier B.V.

Fig. 9.

EVs decorated with RNA nanoparticles increase delivery efficiency and therapeutic effect of siRNA. (A) Schematic of EVs loaded with 3WJ-siRNA and decorated with 3WJ-EGFR_apt; (B) Tumor regression curve of mice bearing NSCLC tumor after treatment. Adapted with permission from [43]. Copyright 2021 Mary Ann Liebert, Inc.; (C) Confocal images of cellular internalization of siRNA and siRNA loaded in RNA decorated EVs (Blue: DAPI; Green: endosome/lysosome; Red: RNA); (D) Schematic of direct membrane fusion mechanism of endosome escape pathway and cellular distribution of fluorescent 3WJ RNA and siRNA after cellular internalization of EVs (Blue: DAPI; Green: Cy3 labeled siRNA; Red: A647 labeled 3WJ RNA). Adapted with permission from [32]. Copyright 2020 Elsevier B.V.