Functionally-interdependent shape-switching nanoparticles with controllable properties.

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.

Controlled Organization of Inorganic Materials Using Biological Molecules for Activating Therapeutic Functionalities.

Precise control over the assembly of biocompatible three-dimensional (3D) nanostructures would allow for programmed interactions within the cellular environment. Nucleic acids can be used as programmable crosslinkers to direct the assembly of quantum dots (QDs) and tuned to demonstrate different interparticle binding strategies. Morphologies of self-assembled QDs are evaluated via gel electrophoresis, transmission electron microscopy, small-angle X-ray scattering, and dissipative particle dynamics simulations, with all results being in good agreement. The controlled assembly of 3D QD organizations is demonstrated in cells via the colocalized emission of multiple assembled QDs, and their immunorecognition is assessed via enzyme-linked immunosorbent assays. RNA interference inducers are also embedded into the interparticle binding strategy to be released in human cells only upon QD assembly, which is demonstrated by specific gene silencing. The programmability and intracellular activity of QD assemblies offer a strategy for nucleic acids to imbue the structure and therapeutic function into the formation of complex networks of nanostructures, while the photoluminescent properties of the material allow for optical tracking in cells in vitro.

Correction: Programmable RNA microstructures for coordinated delivery of siRNAs.

Correction for 'Programmable RNA microstructures for coordinated delivery of siRNAs' by Jaimie Marie Stewart et al., Nanoscale, 2016, 8, 17542-17550.

Programmable RNA microstructures for coordinated delivery of siRNAs.

RNA is a natural multifunctional polymer, and is an essential component in both complex pathways and structures within the cellular environment. For this reason, artificial self-assembling RNA nanostructures are emerging as a powerful tool with broad applications in drug delivery and metabolic pathway regulation. To date, coordinated delivery of functional molecules via programmable RNA assemblies has been primarily done using nanosize RNA scaffolds. However, larger scaffolds could expand existing capabilities for spatial arrangement of ligands, and enable the controlled delivery of highly concentrated molecular loads. Here, we investigate whether micron-size RNA scaffolds can be assembled and further functionalized with different cargos (e.g. various siRNAs and fluorescent tags) for their synchronized delivery to diseased cells. Since known design approaches to build large RNA scaffolds are still underdeveloped, we apply a tiling method widely used in DNA nanotechnology. DNA tiles have been extensively used to build a variety of scalable and modular structures that are easily decorated with other ligands. Here, we adapt a double crossover (DX) DNA tile motif to design de novo DX RNA tiles that assemble and form lattices via programmed sticky end interactions. We optimize assembly protocols to guarantee high yield of RNA lattices. The resulting constructs are robust and modular with respect to the presence of distinct siRNAs and fluorophores. RNA tiles and lattices are successfully transfected in either human breast cancer or prostate cancer cells, where they efficiently knockdown the expression of target genes. Blood serum stability assays indicate that RNA lattices are more resilient to nuclease degradation when compared to individual tiles, thus making them better suited for therapeutic purposes. Overall, because of its design simplicity, we anticipate that this approach will be utilized for a wide range of applications in therapeutic RNA nanotechnology.

Fluorescence Blinking as an Output Signal for Biosensing.

We demonstrate the first biosensing strategy that relies on quantum dot (QD) fluorescence blinking to report the presence of a target molecule. Unlike other biosensors that utilize QDs, our method does not require the analyte to induce any fluorescence intensity or color changes, making it readily applicable to a wide range of target species. Instead, our approach relies on the understanding that blinking, a single particle phenomenon, is obscured when several QDs lie within the detection volume of a confocal microscope. If QDs are engineered to aggregate when they encounter a particular target molecule, the observation of quasi-continuous emission should indicate its presence. As proof of concept, we programmed DNAs to drive rapid isothermal assembly of QDs in the presence of a target strand (oncogene K-ras). The assemblies, confirmed by various gel techniques, contained multiple QDs and were readily distinguished from free QDs by the absence of blinking.