Justin R. Halman

Functionally-interdependent shape-switching nanoparticles with controllable properties.

Abstract We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

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

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.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Label-Free Single-Molecule Thermoscopy Using a Laser-Heated Nanopore

When light is used to excite electronic transitions in a material, nonradiative energy during relaxation is often released in the form of heat. In this work, we show that photoexcitation of a silicon nitride nanopore using a focused visible laser results in efficient localized photothermal heating, which reduces the nearby electrolyte viscosity and increases the ionic conductance. In addition, a strong localized thermal gradient in the pore vicinity is produced, evidenced by finite-element simulations and experimental observation of both ion and DNA thermophoresis. After correcting for thermophoresis, the nanopore current can be used as a nanoscale thermometer, enabling rapid force thermoscopy. We utilize this to probe thermal melting transitions in synthetic and native biomolecules that are heated at the nanopore. Our results on single molecules are validated by correspondence to bulk measurements, which paves the way to various biophysical experiments that require rapid temperature and force control on individual molecules.

Picomolar Fingerprinting of Nucleic Acid Nanoparticles Using Solid-State Nanopores

Nucleic acid nanoparticles (NANPs) are an emerging class of programmable structures with tunable shape and function. Their promise as tools for fundamental biophysics studies, molecular sensing, and therapeutic applications necessitates methods for their detection and characterization at the single-particle level. In this work, we study electrophoretic transport of individual ring-shaped and cube-shaped NANPs through solid-state nanopores. In the optimal nanopore size range, the particles must deform to pass through, which considerably increases their residence time within the pore. Such anomalously long residence times permit detection of picomolar amounts of NANPs when nanopore measurements are carried out at a high transmembrane bias. In the case of a NANP mixture, the type of individual particle passing through nanopores can be efficiently determined from analysis of a single electrical pulse. Molecular dynamics simulations provide insight into the mechanical barrier to transport of the NANPs and corroborate the difference in the signal amplitudes observed for the two types of particles. Our study serves as a basis for label-free analysis of soft programmable-shape nanoparticles.

Reconfigurable Nucleic Acid Materials for Cancer Therapy

According to the WHO, we can expect the rise of around 24 million new cases of cancer per year by around 2030 worldwide, which is a 70% increase from 2012. The molecular key players leading to cancer are heterogeneous in respect to tissue origin and may vary from patient to patient, calling for an individualized approach. Nucleic acid biopolymers (DNA and RNA) lend themselves toward applications in personalized therapeutics with high programmability based on their primary structure of five building blocks as well as biocompatibility based on established roles and functional abilities in vivo. Based on the last decades of advances in synthetic methods and natural functions of RNA, various pathways for the regulation of gene expression and DNA/RNA protein binding pathways have been uncovered, leading to the development of novel nanoparticle formulations with governing design principles. For the translation of such approaches into the clinic, the immunogenicity, strategies for delivery, and integration of mechanisms for conditional activation of therapeutic nucleic acids are explored.

Activation of Multiple Functionalities Through Interacting Nanoparticles

Abstract Nucleic acids are biocompatible, robust, and highly versatile polymers that can be used to design fine-tunable and dynamically responsive nanostructures. In this report, we focus our attention to recently introduced concepts of interdependent, cognate nucleic acid nanoparticles assembly that take advantage of dynamic interactions and consequent shape-switching to trigger the activation of multiple functionalities. Particularly, we discuss re-association of thermodynamically driven complementary nanocubes (“cube” and complementary “anti-cube”) into functional duplexes that do not require toehold interactions or extensive computational design, bringing a new perspective for utility of nucleic acid nanoparticles as a drug carriers, biosensors, and templates for the formation of siRNA duplexes.