RNA Nanotechnology

Cheatsheet Content

### RNA: Overview and Properties - **Central Dogma:** RNA was traditionally seen as an intermediary between DNA and proteins. - Gene sequencing shows ~1-2% of human genome encodes proteins; noncoding RNAs play key regulatory roles. - **Chemical Composition & Structural Diversity:** RNA's unique features allow it to be a biologically functional molecule. - **A-form double helix:** ~3 nm diameter, 11 bp/turn, ~19° base tilt, ~33° base twist angle. - **Nucleobases:** Adenine, Uracil, Guanine, Cytosine. - **Base Pairing:** - **Canonical:** Cytosine-Guanine, Adenine-Uracil. - **Non-canonical:** GU Wobble, G-quartet. - **Structure:** Sugar-phosphate backbone, minor groove, major groove. - **Biological Role:** Cells produce and degrade RNA to carry out functions, making it an ideal biomaterial for interacting with biological components and controlling cell activity. - **Functions in Nature:** Diverse RNA molecules provide new ways to interface with biology (e.g., siRNA, miRNA, riboswitches, ribozymes, aptamers, mRNA). ### RNA Nanotechnology - Harnesses the unique chemical and structural properties of RNA to build functional assemblies. #### Hierarchical Principles - RNA structures fold hierarchically, influenced by: - Base pairing, stacking interactions, electrostatics, tertiary interactions. - Motifs enable programmable nanoarchitectures. - Higher-order assembly driven by: - Coaxial stacking, kissing loops, loop-bulge contacts, sticky ends. #### Computational Modeling in RNA Design - **Secondary Structure Prediction:** ViennaRNA, RNAstructure, NUPACK (MFE structures, base-pair probabilities, ensembles). - **Tertiary Modeling:** Rosetta FARFAR2, RNAComposer (3D geometry, steric constraints, motif placement). - **Coarse-grained Simulation:** oxRNA (dynamics, stability, misfolding pathways). - Modeling guides optimization of folding, energetics, and assembly yield. #### Design Paradigms 1. **Motif-driven Design:** - Uses naturally occurring motifs (e.g., junctions, loops, kissing pairs) as modular building blocks. - Motifs characterized by X-ray, NMR, cryo-EM. - Assembles 2D/3D architectures with predictable geometries. - **RNA Tectonics:** Recombines "TectoRNAs" (solved RNA motifs) to form larger nanoarchitectures. - **RNA Origami:** Single-stranded RNA structures with arrays of anti-parallel helices organized by tertiary motifs. Folds via annealing or co-transcriptional assembly. - **Phi29 Packaging RNA (pRNA):** Re-engineered into RNA nanoparticles via 3-way and 4-way junctions; loop-loop interactions promote higher-order assembly (dimers, trimers, hexamers). 2. **Rational De Novo Design:** - Builds structures using thermodynamics and modeling. - Predicts secondary and tertiary structure computationally. - Enables multi-stranded tiles, nanostars, origami, nanoparticles. - Allows precise control of symmetry, valency, rigidity, and interaction energy. - **RNA Tiles:** Structural elements designed de novo, guided by computational tools to program predictable architectures. - **Nanostars:** Designed de novo using RNA folding and thermodynamics to program predictable multivalent junctions. - **Integration:** Natural RNA motifs offer validated modules, while de novo design expands possibilities. Integration enables predictable folding, programmable valency, controlled topology, and modular functionalization. #### Building Supramolecular Architectures - Arises from multivalent interactions beyond single motifs or tiles. - Higher-order structures emerge from: sticky-end hybridization, kissing-loop networks, junction-driven scaffolding, long-range tertiary interactions. - Enables construction of large 2D/3D lattices, nanotubes, polyhedra, and condensates. - Design must consider stoichiometry, mass action, ionic environment, and assembly pathway. ### RNA Synthesis - RNA nanostructure design is constrained by how RNA is produced. - Methods determine feasible lengths, purity, and chemical modification options. #### Solid-Phase Synthesis - Stepwise chemical synthesis where RNA/DNA chain grows on a solid support (glass, resin, silica). - **Considerations:** - Length limited by stepwise coupling yield. - Enables precise chemical modifications (e.g., 2’-OMe, 2’-F, PS, fluorophores, biotin). - Practical length limit for high-purity RNA is ~80-120 NT. - Synthetic oligonucleotides obtained from commercial providers (e.g., IDT, Twist). #### In Vitro RNA Transcription - Generates RNA strands from a DNA template, enabling high-yield RNA production. - **Components:** DNA template, RNA polymerase (e.g., T7, T3, SP6), rNTPs, buffer and Mg²⁺. - **Reaction Conditions:** Transcription proceeds at ~37 °C. #### Transcriptional Assembly vs. Thermal Annealing - **Annealing-based Assembly:** - Multi-stranded RNAs mixed, heated, and slowly cooled. - Better approximates thermodynamic equilibrium structure. - Kinetic traps can occur for complex architectures. - Best for tiles, nanostars, and RNA nanoparticles. - **Thermodynamic control.** - **Transcriptional Assembly:** - Folding occurs co-transcriptionally. - Structure formation steered by elongation order (kinetic control). - Enables long single-stranded architectures (e.g., RNA origami). - One-pot assembly directly from DNA template. - **Kinetic control.** - Choice affects yield, misfolding, stoichiometry, and scalability. ### Thermodynamics of RNA Folding & Assembly - RNA folding governed by nearest-neighbor energetics and ionic strength. - Structure stability determined by ΔG°, ΔH°, ΔS° of stacked base pairs. - Mg²⁺ strongly stabilizes tertiary structure and increases Tm. - Tertiary motifs complicate prediction. - **Design Implications:** - Sticky ends should have Tm ≥ assembly temperature +5-10°C. - Avoid competing secondary structures. - Consider concentration-dependent assembly (mass action). #### Avoiding RNA Misfolding - RNA folding landscape is rugged with many metastable states. - Co-transcriptional folding can trap early hairpins, blocking correct topology. - **Common sources of misfolding:** unintended hairpins, partial/spurious duplexes, overly stable local structures. - **Design strategies:** - Minimize undesired structures using ensemble diversity metrics. - Delay formation of stable hairpins (place them later in sequence). - Use weak stems that dissolve after long-range contacts form. - Ensure tertiary interactions occur before misfolding pathways dominate. #### Assembly Kinetics and Mass Action - Supramolecular assembly follows mass-action kinetics. - At equilibrium, $K_{eq} = k_{on} / k_{off} = e^{-\Delta G^\circ / RT}$. - Valency controls assembly behavior. - Assembly yield increases with: more negative sticky-end ΔG°, higher strand concentration, Mg²⁺. - **Design Principle:** Control stoichiometry to avoid aggregation or incomplete structures. ### Functional RNA Motifs & Applications - **siRNA (small interfering RNA):** Gene silencing. - **microRNA (miRNA):** Post-transcriptional regulation. - **Riboswitches:** Ligand-responsive regulation. - **Ribozymes:** Catalysis. - **RNA Aptamers:** Molecular recognition. - **mRNA:** Encodes protein. #### RNA Interference (RNAi) - Conserved process where double-stranded RNA triggers sequence-specific silencing of target RNAs. - **siRNA (~21-23 NT):** Pairs with full complementarity, induces target cleavage. - **miRNA (~19-25 NT):** Pairs with partial complementarity, represses translation or destabilizes target. #### Riboswitches - Cis-acting regulatory RNAs with aptamer domain and expression platform. - Ligand binding induces conformational change, toggling gene expression. - Regulate transcription, translation, or splicing. #### Ribozymes - Catalytic RNAs that accelerate specific biochemical reactions (cleave or ligate RNA). - **Natural classes:** Hammerhead, Twister, Hepatitis Delta Virus (HDV). #### RNA Aptamers - Structured RNAs that bind targets with high affinity and specificity. - Enable targeted delivery, sensing, or molecular antagonism. - Generated by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). #### Functional RNA Tiles - Functional modules can be embedded within RNA tiles without disrupting geometry. - Tiles can present dicer substrate RNAs for siRNA release. - Modular design enables programmable patterns of RNA output. - Functionalized tiles retain assembly ability (1D, 2D, 3D). - **Therapeutic Potential:** Demonstrated gene silencing (e.g., PLK1 in prostate cancer cells, GFP in breast cancer cells). - **Stability:** Lattices provide increased stability in human blood serum compared to individual tiles. #### Fluorogenic RNA Aptamers - Bind fluorogens, causing fluorescence (e.g., Corn, Orange Broccoli, Red Broccoli aptamers binding DFHO). - Often involve G-quartet/G-quadruplex structures. - RNA nanostars can be modified with aptamers for fluorescent condensates to recruit molecules. ### Challenges in RNA Nanotechnology - Predicting folding and assembly. - Stability. - RNA immunogenicity. - Large scale synthesis. - Delivery constraints. - Off-target effects. #### Delivery and Cellular Barriers - **Biological Barriers:** Degradation by RNases, limited cellular uptake of naked RNA, endosomal entrapment, innate immune sensing. - **Delivery Platforms:** Lipid nanoparticles, RNA-polymer complexes, aptamer-guided targeting. - **Engineering Considerations:** Charge, particle size, chemical modifications to reduce innate immune activation. #### Innate Immune Recognition of RNA Nanostructures - **Important Receptors:** - RIG-I: short dsRNA with 5’-triphosphate (5’-ppp). - MDA5: long dsRNA. - TLRs (3, 7, 8): endosomal sensing of dsRNA or ssRNA. - **Immunostimulatory RNA Features:** Long dsRNA (>300 bp), 5’-ppp, GU-rich or AU-rich motifs. - **Design Implications:** Chemical modifications (2’-OMe, 2’-F, Ψ) reduce innate sensing. Consider intracellular localization and delivery route. #### Modifying RNA for Robustness and Biocompatibility - Over 170 chemical modifications identified in native RNA. - Modifications incorporated by chemical or enzymatic methods to improve stability, immunocompatibility, pharmacokinetics, and folding behavior. - **Phosphorothioate:** Nuclease resistance. - **2’-F and 2’-OCH₃:** Bias C3’-endo sugar pucker, increase stability, reduce immune sensing. - **m6A, m5C, Ψ, s2U:** Tune hydrogen bonding, stacking, and innate immune recognition. #### Future of RNA Nanotechnology - Future progress relies on accurate folding prediction, scalable and robust RNA synthesis, immune-compatible architectures, and advanced delivery strategies to enable engineered RNA assemblies to function within living systems.