Daniel L. Jasinski

RNA as a Boiling-Resistant Anionic Polymer Material To Build Robust Structures with Defined Shape and Stoichiometry

RNA is a polyribonucleic acid belonging to a special class of anionic polymers, holding a unique property of self-assembly that is controllable in the construction of structures with defined size, shape, and stoichiometry. We report here the use of RNA as polymers to fabricate boiling-resistant triangular nanoscaffolds, which were used to construct hexagons and patterned hexagonal arrays. The RNA triangular scaffolds demonstrated promising potential to construct fluorogenic probes and therapeutic agents as functionalization with siRNA, ribozyme, folate, and fluorogenic RNA aptamers revealed independent functional activity of each RNA moiety. The ribozyme was able to cleave hepatitis genomic RNA fragments, the siRNA silenced the target genes, and all fluorogenic RNA aptamers retained their fluorescence emission property. The creation of boiling-temperature-resistant RNA nanoparticles opens a new dimension of RNA as a special polymer, feasible in industrial and nanotechnological applications.

Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles

Modulation of immune response is important in cancer immunotherapy, vaccine adjuvant development and inflammatory or immune disease therapy. Here we report the development of new immunomodulators via control of shape transition among RNA triangle, square and pentagon. Changing one RNA strand in polygons automatically induced the stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares and pentagons. When immunological adjuvants were incorporated, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced in vitro and in animals up to 100-fold, while RNA polygon controls induced unnoticeable effect. The RNA nanoparticles were delivered to macrophages specifically. The degree of immunostimulation greatly depended on the size, shape and number of the payload per nanoparticles. Stronger immune response was observed when the number of adjuvants per polygon was increased, demonstrating the advantage of shape transition from triangle to pentagon.

Physicochemically Tunable Polyfunctionalized RNA Square Architecture with Fluorogenic and Ribozymatic Properties

Recent advances in RNA nanotechnology allow the rational design of various nanoarchitectures. Previous methods utilized conserved angles from natural RNA motifs to form geometries with specific sizes. However, the feasibility of producing RNA architecture with variable sizes using native motifs featuring fixed sizes and angles is limited. It would be advantageous to display RNA nanoparticles of diverse shape and size derived from a given primary sequence. Here, we report an approach to construct RNA nanoparticles with tunable size and stability. Multifunctional RNA squares with a 90° angle were constructed by tuning the 60° angle of the three-way junction (3WJ) motif from the packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor. The physicochemical properties and size of the RNA square were also easily tuned by modulating the “core” strand and adjusting the length of the sides of the square via predictable design. Squares of 5, 10, and 20 nm were constructed, each showing diverse thermodynamic and chemical stabilities. Four “arms” extending from the corners of the square were used to incorporate siRNA, ribozyme, and fluorogenic RNA motifs. Unique intramolecular contact using the pre-existing intricacy of the 3WJ avoids relatively weaker intermolecular interactions via kissing loops or sticky ends. Utilizing the 3WJ motif, we have employed a modular design technique to construct variable-size RNA squares with controllable properties and functionalities for diverse and versatile applications with engineering, pharmaceutical, and medical potential. This technique for simple design to finely tune physicochemical properties adds a new angle to RNA nanotechnology.

Advancement of the Emerging Field of RNA Nanotechnology

The field of RNA nanotechnology has advanced rapidly during the past decade. A variety of programmable RNA nanoparticles with defined shape, size, and stoichiometry have been developed for diverse applications in nanobiotechnology. The rising popularity of RNA nanoparticles is due to a number of factors: (1) removing the concern of RNA degradation in vitro and in vivo by introducing chemical modification into nucleotides without significant alteration of the RNA property in folding and self-assembly; (2) confirming the concept that RNA displays very high thermodynamic stability and is suitable for in vivo trafficking and other applications; (3) obtaining the knowledge to tune the immunogenic properties of synthetic RNA constructs for in vivo applications; (4) increased understanding of the 4D structure and intermolecular interaction of RNA molecules; (5) developing methods to control shape, size, and stoichiometry of RNA nanoparticles; (6) increasing knowledge of regulation and processing functions of RNA in cells; (7) decreasing cost of RNA production by biological and chemical synthesis; and (8) proving the concept that RNA is a safe and specific therapeutic modality for cancer and other diseases with little or no accumulation in vital organs. Other applications of RNA nanotechnology, such as adapting them to construct 2D, 3D, and 4D structures for use in tissue engineering, biosensing, resistive biomemory, and potential computer logic gate modules, have stimulated the interest of the scientific community. This review aims to outline the current state of the art of RNA nanoparticles as programmable smart complexes and offers perspectives on the promising avenues of research in this fast-growing field.

Fabrication of RNA 3D Nanoprisms for Loading and Protection of Small RNAs and Model Drugs

Constructing containers with defined shape and size to load and protect therapeutics and subsequently control their release in the human body has long been a dream. The fabrication of 3D RNA prisms, characterized by atomic force microscopy, cryo-electron microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis, is reported for the loading and protection of small molecules, proteins, small RNA molecules, and their controlled release.

Large Scale Purification of RNA Nanoparticles by Preparative Ultracentrifugation

Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.

Directional mechanical stability of Bacteriophage φ29 motor’s 3WJ-pRNA: Extraordinary robustness along portal axis

φ29 motor’s three-way junction serves as an effective connecting rod through its superb rigidity in the coaxial direction. The molecular motor exploited by bacteriophage φ29 to pack DNA into its capsid is regarded as one of the most powerful mechanical devices present in viral, bacterial, and eukaryotic systems alike. Acting as a linker element, a prohead RNA (pRNA) effectively joins the connector and ATPase (adenosine triphosphatase) components of the φ29 motor. During DNA packing, this pRNA needs to withstand enormous strain along the capsid’s portal axis—how this remarkable stability is achieved remains to be elucidated. We investigate the mechanical properties of the φ29 motor’s three-way junction (3WJ)–pRNA using a combined steered molecular dynamics and atomic force spectroscopy approach. The 3WJ exhibits strong resistance to stretching along its coaxial helices, demonstrating its super structural robustness. This resistance disappears, however, when external forces are applied to the transverse directions. From a molecular standpoint, we demonstrate that this direction-dependent stability can be attributed to two Mg clamps that cooperate and generate mechanical resistance in the pRNA’s coaxial direction. Our results suggest that the asymmetric nature of the 3WJ’s mechanical stability is entwined with its biological function: Enhanced rigidity along the portal axis is likely essential to withstand the strain caused by DNA condensation, and flexibility in other directions should aid in the assembly of the pRNA and its association with other motor components.

Evaluation of Thermal Stability of RNA Nanoparticles by Temperature Gradient Gel Electrophoresis (TGGE) in Native Condition

Temperature gradient gel electrophoresis (TGGE) is a powerful tool used to analyze the thermal stabilities of nucleic acids. While TGGE is a decades-old technique, it has recently gained favor in the field of RNA nanotechnology, notably in assessing the thermal stabilities of RNA nanoparticles (NPs). With TGGE, an electrical current and a linear temperature gradient are applied simultaneously to NP-loaded polyacrylamide gel, separating the negatively charged NPs based on their thermal behavior (a more stable RNA complex will remain intact through higher temperature ranges). The linear temperature gradient can be set either perpendicular or parallel to the electrical current, as either will make the NPs undergo a transition from native to denatured conformations. Often, the melting transition is influenced by sequence variations, secondary/tertiary structures, concentrations, and external factors such as the presence of a denaturing agent (e.g., urea), the presence of monovalent or divalent metal ions, and the pH of the solvent. In this chapter, we describe the experimental setup and the analysis of the thermal stability of RNA NPs in native conditions using a modified version of a commercially available TGGE system.

Simple Method for Constructing RNA Triangle, Square, Pentagon by Tuning Interior RNA 3WJ Angle from 60° to 90° or 108°.

Precise shape control of architectures at the nanometer scale is an intriguing but extremely challenging facet. RNA has recently emerged as a unique material and thermostable building block for use in nanoparticle construction. Here, we describe a simple method from design to synthesis of RNA triangle, square, and pentagon by stretching RNA 3WJ native angle from 60° to 90° and 108°, using the three-way junction (3WJ) of the pRNA from bacteriophage phi29 dsDNA packaging motor. These methods for the construction of elegant polygons can be applied to other RNA building blocks including the utilization and application of RNA 4-way, 5-way, and other multi-way junctions.

RNA nanoparticle distribution and clearance in the eye after subconjunctival injection with and without thermosensitive hydrogels

Abstract Thermodynamically and chemically stable RNA nanoparticles derived from the three‐way junction (3WJ) of the pRNA from bacteriophage phi29 DNA packaging motor were examined previously for ocular delivery. It was reported that, after subconjunctival injection, RNA nanoparticles with tri‐way shape entered the corneal cells but not the retinal cells, whereas particle with four‐way shape entered both corneal and retinal cells. The present study evaluated ocular delivery of RNA nanoparticles with various shapes and sizes, and assessed the effect of thermosensitive hydrogels (poly(lactic‐co‐glycolic acid)‐b‐poly(ethylene glycol)‐b‐poly(lactic‐co‐glycolic acid); PLGA‐PEG‐PLGA) for increasing the retention of RNA nanoparticles in the eye. Fluorescence imaging of mouse eyes and fluorescence microscopy of dissected eye tissues from the conjunctiva, cornea, retina, and sclera were performed to determine the distribution and clearance of the nanoparticles in the eyes after subconjunctival injection in vivo. RNA nanoparticles entered the cells of the conjunctiva, cornea, retina, and sclera after subconjunctival delivery. The clearance of RNA pentagon was slower than both RNA square and triangle of the same designed edge length (10 nm) in the eye, and the clearance of RNA squares of the longer edge lengths (10 and 20 nm) was slower than RNA square of the shorter edge length (5 nm), thus indicating that the size could affect ocular pharmacokinetics of the nanoparticles. At 24 h after the injection, approximately 6–10% of the fluorescence signal from the larger nanoparticles in the study (RNA square of 20 nm edge length and RNA pentagon of 10 nm edge length) remained in the eye, and up to 70% of the retinal cells contained the nanoparticles. The results suggest that the larger nanoparticles were “gulped” in conjunctival, corneal, retinal, and scleral cells, similar to the behavior observed in macrophages. Additionally, the combination of RNA nanoparticles with the thermosensitive polymers increased the retention of the nanoparticles in the eye. Graphical abstract Figure. No Caption available.