Functionally-interdependent shape switching nucleic acid nanoparticles
Inventors
Shapiro, Bruce Allen • Afonin, Kirill Andreevich • Bindewald, Eckart H. U. • Viard, Mathias D. • Kasprzak, Wojciech • Dobrovolskaia, Marina A. • Halman, Justin R.
Assignees
University of North Carolina at Charlotte • US Department of Health and Human Services
Publication Number
US-11512313-B2
Publication Date
2022-11-29
Expiration Date
2038-04-03
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Abstract
The description provides a molecular switch comprising at least two nanoparticles, wherein a first nanoparticle comprises DNA and/or RNA oligonucleotides, and a second nanoparticle which is complementary to the first nanoparticle comprises reverse complementary DNA and/or RNA oligonucleotides of the first nanoparticle; wherein the complementary nanoparticles interact under physiological conditions leading to thermodynamically driven conformational changes in the first and second nanoparticles leading to their re-association to release one or more duplexes comprising said DNA and/or RNA oligonucleotides and the reverse complementary DNA and/or RNA oligonucleotides, and wherein the nanoparticles are not rings and have no single stranded toeholds.
Core Innovation
The invention relates to a system comprising at least two complementary nucleic acid nanoparticles, each formed from DNA and/or RNA oligonucleotides. A first nanoparticle comprises DNA and/or RNA oligonucleotides, and a second nanoparticle complementary to the first comprises reverse complementary DNA and/or RNA oligonucleotides. These complementary nanoparticles interact under physiological conditions resulting in thermodynamically driven conformational changes leading to re-association and simultaneous disassembly of the initial nanoparticles. This process leads to the release of multiple functional duplexes comprising DNA and/or RNA strands from respective nanoparticles, thereby activating latent functionalities that were initially divided between the complementary particles.
The background outlines challenges in nanobiology related to efficient delivery of therapeutic agents, such as siRNAs, for diagnosis and treatment across various diseases. Existing RNA and DNA nanoparticles show programmability and biocompatibility but often require multiple components, toeholds, or complex designs to activate functionalities, often limited to in vitro settings, and face issues with synthesis, targeting, immunogenicity, and stability in vivo.
The innovation solves these problems by introducing functionally interdependent shape-switching nucleic acid nanoparticles designed simply by taking reverse complements (scaffold and anti-scaffold; cube and anti-cube), which upon interaction undergo shape switching without the need for single-stranded toeholds or multiple components. This approach activates multiple functionalities simultaneously from only two complementary nanoparticles under physiological conditions, allowing for simplified design, enhanced activation control, improved stability, tunable immunogenicity, and potential for therapeutic applications in living systems.
Claims Coverage
The claims encompass one main inventive system with multiple inventive features leveraging complementary nucleic acid nanoparticles for multiplexed latent functionality activation.
Complementary nucleic acid nanoparticle system activating multiple functionalities
A system comprising at least two complementary nanoparticles, each formed from DNA and/or RNA oligonucleotides, wherein the complementary pairs interact under physiological conditions to undergo thermodynamically driven conformational changes that simultaneously disassemble initial nanoparticles and form multiple functional duplexes without the need for single stranded toeholds or ring structures.
Functionalities selectable from RNAi, transcription, gene silencing, optical response, RNA cube formation, and split aptamer
Nanoparticles are capable of containing latent functionalities selected from RNA interference, transcriptional activity, gene silencing, optical detection (e.g., FRET), RNA cube assembly, and split aptamer reconstitution, which become activated upon nanoparticle interaction and duplex formation.
Nanoparticle shapes including cubes, tubes, and triangles
The invention includes nanoparticles in shapes such as cubes, tubes, and triangles, but explicitly excludes ring-shaped nanoparticles because rings do not re-associate to form functional duplexes.
Ratio of DNA to RNA controlling physical and immunological properties
The ratio of DNA and RNA strands in the nanoparticles controls immunostimulatory activity, thermodynamic stability, nuclease resistance, re-association kinetics, and production cost, enabling fine-tuning of nanoparticle properties.
Complementary nanoparticles comprising six stranded cubes and anti-cubes
Complementary nanoparticles may be six-stranded cubes and anti-cubes constructed from DNA, RNA, or mixtures thereof in different ratios to tailor functionality and properties.
Nanoparticles with arm sequences providing multiple functionalities
The six oligonucleotide strands of the cubes may include arm sequences that comprise split RNAi agents or other functionalities, enabling multiple distinct target gene sequences to be addressed by the nanoparticles.
Compositions including lipid-based delivery systems
The nanoparticles may be composed with lipid-based delivery systems (e.g., Lipofactamine 2000) to facilitate delivery and immune response induction post-delivery.
The patent claims cover a complementary nucleic acid nanoparticle system of various shapes, particularly six-stranded cubes and anti-cubes, designed to undergo toehold-free interaction under physiological conditions and simultaneous disassembly with paired duplex formation. This enables conditional activation of multiple split latent functionalities such as RNAi, transcription, optical signals, and aptamer reassembly. The system allows tunable physical and immunological properties by adjusting DNA/RNA ratios and supports delivery by lipid systems.
Stated Advantages
Activates multiple latent functionalities simultaneously using only two complementary nanoparticles without requiring toeholds.
Simplified design principles by using reverse complements and avoiding complex computer-assisted design.
Ability to fine-tune immunogenicity, thermal stability, nuclease resistance, and re-association rate by varying DNA to RNA strand ratios.
Improved in vivo applicability through conditionally activated RNAi, transcriptional activation, optical detection, and aptamer reassembly.
Potential for cost-effective manufacturing and scalable production due to simplified nanoparticle assembly.
Documented Applications
Delivery of therapeutic and diagnostic functionalities such as siRNAs for gene silencing, RNAi therapeutics, and targeted gene knockdown.
Co-transcriptional production of RNA nanoparticles inside cells by activating transcription from complementary DNA nanoparticle duplexes.
Activation of optical reporters, such as FRET pairs and fluorescent RNA aptamers, allowing intracellular detection of nanoparticle interactions.
Use as multi-functional drug delivery systems for treating viral infections including HIV, various cancers, and neoplasms.
Application in killing pathogen-infected cells and reducing pathogenic burden in subjects infected by viruses, bacteria, fungi, or parasites.
Potential use as vaccine adjuvants due to tunable immune stimulatory properties.
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