HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5'cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5'cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3'UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson-Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson-Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Dynamic bulge nucleotides in the KSHV PAN ENE triple helix provide a unique binding platform for small molecule ligands.

Cellular and virus-coded long non-coding (lnc) RNAs support multiple roles related to biological and pathological processes. Several lncRNAs sequester their 3' termini to evade cellular degradation machinery, thereby supporting disease progression. An intramolecular triplex involving the lncRNA 3' terminus, the element for nuclear expression (ENE), stabilizes RNA transcripts and promotes persistent function. Therefore, such ENE triplexes, as presented here in Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) lncRNA, represent targets for therapeutic development. Towards identifying novel ligands targeting the PAN ENE triplex, we screened a library of immobilized small molecules and identified several triplex-binding chemotypes, the tightest of which exhibits micromolar binding affinity. Combined biophysical, biochemical, and computational strategies localized ligand binding to a platform created near a dinucleotide bulge at the base of the triplex. Crystal structures of apo (3.3 Å) and ligand-soaked (2.5 Å) ENE triplexes, which include a stabilizing basal duplex, indicate significant local structural rearrangements within this dinucleotide bulge. MD simulations and a modified nucleoside analog interference technique corroborate the role of the bulge and the base of the triplex in ligand binding. Together with recently discovered small molecules that reduce nuclear MALAT1 lncRNA levels by engaging its ENE triplex, our data supports the potential of targeting RNA triplexes with small molecules.

Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target.

The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.

Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway.

Programmed -1 ribosomal frameshift (-1 PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. Here we describe a -1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co-receptor. CCR5 mRNA-mediated -1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA-miRNA interaction suggests that formation of a triplex RNA structure stimulates -1 PRF. A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional -1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells.

RiboSketch: versatile visualization of multi-stranded RNA and DNA secondary structure.

Summary: Creating clear, visually pleasing 2D depictions of RNA and DNA strands and their interactions is important to facilitate and communicate insights related to nucleic acid structure. Here we present RiboSketch, a secondary structure image production application that enables the visualization of multistranded structures via layout algorithms, comprehensive editing capabilities, and a multitude of simulation modes. These interactive features allow RiboSketch to create publication quality diagrams for structures with a wide range of composition, size and complexity. The program may be run in any web browser without the need for installation, or as a standalone Java application. Availability and implementation: https://rnastructure.cancer.gov/ribosketch.

RNA2Drawer: geometrically strict drawing of nucleic acid structures with graphical structure editing and highlighting of complementary subsequences.

RNA structure prediction programs remain imperfect and many substructures are still identified by manual exploration, which is most efficiently conducted within an RNA structure drawing program. However, most nucleic acid structure drawing programs have limited capability for structure modification (i.e., breaking and forming new bonds between bases), often requiring that the structure notation be textually edited. RNA2Drawer was developed to allow for graphical structure editing while maintaining the geometry of a drawing (e.g., ellipsoid loops, stems with evenly stacked base pairs) throughout structural changes and manual adjustments to the layout by the user. In addition, the program allows for annotations such as colouring and circling of bases and drawing of tertiary interactions (e.g., pseudoknots). RNA2Drawer can also draw commonly desired elements such as an optionally flattened outermost loop and assists structure editing by automatically highlighting complementary subsequences, which facilitates the discovery of potentially new and alternative pairings, particularly tertiary pairings over long-distances, which are biologically critical in the genomes of many RNA viruses and cannot be accurately predicted by current structure prediction programs. Additionally, RNA2Drawer outputs drawings either as PNG files, or as PPTX and SVG files, such that every object of a drawing (e.g., bases, bonds) is an individual PPTX or SVG object, allowing for further manipulation in Microsoft PowerPoint or a vector graphics editor such as Adobe Illustrator. PowerPoint is the standard for presentations and is often used to create figures for publications, and RNA2Drawer is the first program to export drawings as PPTX files.

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.

Dynamic Behavior of RNA Nanoparticles Analyzed by AFM on a Mica/Air Interface.

RNA is an attractive biopolymer for engineering self-assembling materials suitable for biomedical applications. Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduce gaps into the double-stranded regions of the RNA rings. Molecular dynamic simulations are used to assess the dynamic behavior and the changes in the flexibility of novel designs. The changes suggested by simulations, however, cannot be clearly confirmed by the conventional techniques such as nondenaturing polyacrylamide gel electrophoresis (native-PAGE) and dynamic light scattering (DLS). Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of new assemblies. To address these deficiencies, we introduce a computer-assisted quantification strategy. This strategy is based on an algorithmic atomic force microscopy (AFM)-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validate this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis are anticipated to provide a broad spectrum approach for the future development of nucleic acid-based nanotechnology.

RNA⁻Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics.

Cells frequently simultaneously express RNAs and cognate antisense transcripts without necessarily leading to the formation of RNA duplexes. Here, we present a novel transcriptome-wide experimental approach to ascertain the presence of accessible double-stranded RNA structures based on sequencing of RNA fragments longer than 18 nucleotides that were not degraded by single-strand cutting nucleases. We applied this approach to four different cell lines with respect to three different treatments (native cell lysate, removal of proteins, and removal of ribosomal RNA and proteins). We found that long accessible RNA duplexes were largely absent in native cell lysates, while the number of RNA duplexes was dramatically higher when proteins were removed. The majority of RNA duplexes involved ribosomal transcripts. The duplex formation between different non-ribosomal transcripts appears to be largely of a stochastic nature. These results suggest that cells are-via RNA-binding proteins-mostly devoid of long RNA duplexes, leading to low "noise" in the molecular patterns that are utilized by the innate immune system. These findings have implications for the design of RNA interference (RNAi)-based therapeutics by imposing structural constraints on designed RNA complexes that are intended to have specific properties with respect to Dicer cleavage and target gene downregulation.

Combined single molecule experimental and computational approaches for understanding the unfolding pathway of a viral translation enhancer that participates in a conformational switch.

How plus-strand [+]RNA virus genomes transition from translation templates to replication templates is a matter of much speculation. We have previously proposed that, for Turnip crinkle virus, binding of the encoded RNA-dependent RNA polymerase (RdRp) to the 3'UTR of the [+]RNA template promotes a regional wide-spread conformational switch to an alternative structure that disassembles the cap-independent translation enhancer (CITE) in the 3'UTR. The active 3'CITE folds into a tRNA-like T-shaped structure (TSS) that binds to 80S ribosomes and 60S subunits in the P-site. In this Point-of-View, we discuss the history of our research on the TSS and our recent report combining coarse level single molecule force spectroscopy (optical tweezers) with fine-grain computer simulations of this experimental process and biochemical approaches to obtain a detailed understanding of how RdRp binding in the TSS vicinity might lead to an extensive rearrangement of the RNA structure.

Co-transcriptional production of RNA-DNA hybrids for simultaneous release of multiple split functionalities.

Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA-DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA-RNA, RNA-DNA and DNA-DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.

The 3' untranslated region of Pea Enation Mosaic Virus contains two T-shaped, ribosome-binding, cap-independent translation enhancers.

Many plant viruses without 5' caps or 3' poly(A) tails contain 3' proximal, cap-independent translation enhancers (3'CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3'CITEs participate in a long-distance kissing-loop interaction with a 5' proximal hairpin to deliver ribosomal subunits to the 5' end for translation initiation. Pea Enation Mosaic Virus (PEMV) contains two adjacent 3'CITEs in the center of its 703-nucleotide 3' untranslated region (3'UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3'CITE located near the 3' terminus. This 3'CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3'CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3'TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3'TSS contains an apical loop capable of forming a kissing-loop interaction with a 5' proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3'TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3'UTR in vitro, deleting the normally required kl-TSS and PTE 3'CITEs and placing the kl-H and 3'TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3'CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3'CITE identification. Importance: The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3' cap-independent translation enhancers (3'CITEs) to effectively compete with ongoing host translation. Since only single 3'CITEs have been identified for the vast majority of individual viruses, it is widely accepted that this is sufficient for a virus's translational needs. Pea Enation Mosaic Virus possesses a ribosome-binding 3'CITE that can connect to the 5' end through an RNA-RNA interaction and an adjacent eukaryotic translation initiation factor 4E-binding 3'CITE. We report the identification of a third 3'CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3' and 5' ends. Although both ribosome-binding 3'CITEs are critical for virus accumulation in vivo, only the CITE closest to the termination codon of a reporter open reading frame is active, suggesting that artificial constructs used for 3'CITE identification may underestimate the number of CITEs that participate in translation.

In silico design and enzymatic synthesis of functional RNA nanoparticles.

CONSPECTUS: The use of RNAs as scaffolds for biomedical applications has several advantages compared with other existing nanomaterials. These include (i) programmability, (ii) precise control over folding and self-assembly, (iii) natural functionalities as exemplified by ribozymes, riboswitches, RNAi, editing, splicing, and inherent translation and transcription control mechanisms, (iv) biocompatibility, (v) relatively low immune response, and (vi) relatively low cost and ease of production. We have tapped into several of these properties and functionalities to construct RNA-based functional nanoparticles (RNA NPs). In several cases, the structural core and the functional components of the NPs are inherent in the same construct. This permits control over the spatial disposition of the components, intracellular availability, and precise stoichiometry. To enable the generation of RNA NPs, a pipeline is being developed. On one end, it encompasses the rational design and various computational schemes that promote design of the RNA-based nanoconstructs, ultimately producing a set of sequences consisting of RNA or RNA-DNA hybrids, which can assemble into the designed construct. On the other end of the pipeline is an experimental component, which takes the produced sequences and uses them to initialize and characterize their proper assembly and then test the resulting RNA NPs for their function and delivery in cell culture and animal models. An important aspect of this pipeline is the feedback that constantly occurs between the computational and the experimental parts, which synergizes the refinement of both the algorithmic methodologies and the experimental protocols. The utility of this approach is depicted by the several examples described in this Account (nanocubes, nanorings, and RNA-DNA hybrids). Of particular interest, from the computational viewpoint, is that in most cases, first a three-dimensional representation of the assembly is produced, and only then are algorithms applied to generate the sequences that will assemble into the designated three-dimensional construct. This is opposite to the usual practice of predicting RNA structures from a given sequence, that is, the RNA folding problem. To be considered is the generation of sequences that upon assembly have the proper intra- or interstrand interactions (or both). Of particular interest from the experimental point of view is the determination and characterization of the proper thermodynamic, kinetic, functionality, and delivery protocols. Assembly of RNA NPs from individual single-stranded RNAs can be accomplished by one-pot techniques under the proper thermal and buffer conditions or, potentially more interestingly, by the use of various RNA polymerases that can promote the formation of RNA NPs cotransciptionally from specifically designed DNA templates. Also of importance is the delivery of the RNA NPs to the cells of interest in vitro or in vivo. Nonmodified RNAs rapidly degrade in blood serum and have difficulties crossing biological membranes due to their negative charge. These problems can be overcome by using, for example, polycationic lipid-based carriers. Our work involves the use of bolaamphiphiles, which are amphipathic compounds with positively charged hydrophilic head groups at each end connected by a hydrophobic chain. We have correlated results from molecular dynamics computations with various experiments to understand the characteristics of such delivery agents.

Multifunctional RNA nanoparticles.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Application of Molecular Dynamics to Expand Docking Program's Exploratory Capabilities and to Evaluate Its Predictions.

Recognition of the growing importance of RNA as a target for therapeutic or diagnostic ligands brings the importance of computational predictions of docking poses to such receptors to the forefront. Most docking programs have been optimized for protein targets, based on a relatively rich pool of known docked protein structures. Unfortunately, despite progress, numbers of known docked RNA complexes are low and the accuracy of the computational predictions trained on those inadequate samples lags behind that achieved for proteins. Compared to proteins, RNA structures generally have fewer docking pockets, have less diverse electrostatic surfaces, and are more flexible, raising the possibility of producing only transiently available good docking targets. We are presenting a docking prediction protocol that adds molecular dynamics simulations before and after the actual docking in order to explore the conformational space of the target RNA and then to reevaluate the stability of the predicted RNA-ligand complex. In this way we are attempting to overcome important limitations of the docking programs: the rigid (fully or mostly) target structure and imperfect nature of the docking scoring functions.

Conformational Dynamics of the Hepatitis B Virus Pre-genomic RNA on Multiple Time Scales: Implications for Viral Replication.

Human hepatitis B virus (HBV) replication is initiated by the binding of the viral polymerase (P) to epsilon (ε), an ≈85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 5'-end of the pre-genomic RNA (pgRNA). This interaction triggers P and pgRNA packaging and protein-primed reverse transcription and is therefore an attractive therapeutic target. Our recent nuclear magnetic resonance (NMR) structure of ε provides a useful starting point toward a detailed understanding of HBV replication, and hints at the functional importance of ε dynamics. Here, we present a detailed description of ε motions on the ps to ns and µs to ms time scales by NMR spin relaxation and relaxation dispersion, respectively. We also carried out molecular dynamics simulations to provide additional insight into ε conformational dynamics. These data outline a series of complex motions on multiple time scales within ε. Moreover, these motions occur in mostly conserved nucleotides from structural regions (i.e., priming loop, pseudo-triloop, and U43 bulge) that biochemical and mutational studies have shown to be essential for P binding, P-pgRNA packaging, protein-priming, and DNA synthesis. Taken together, our work implicates RNA dynamics as an integral feature that governs HBV replication.

Structural insights of the conserved "priming loop" of hepatitis B virus pre-genomic RNA.

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral polymerase with a cis-acting regulatory signal, designated epsilon (ε), located at the 5'-end of its pre-genomic RNA (pgRNA). Binding of polymerase to ε is also necessary for pgRNA encapsidation. While the mechanistic basis of this interaction remains elusive, mutagenesis studies suggest its internal 6-nt "priming loop" provides an important structural contribution. ε might therefore be considered a promising target for small molecule interventions to complement current nucleoside-analog based anti-HBV therapies. An ideal prerequisite to any RNA-directed small molecule strategy would be a detailed structural description of this important element. Herein, we present a solution NMR structure for HBV ε which, in combination with molecular dynamics and docking simulations, reports on a flexible ligand "pocket", reminiscent of those observed in proteins. We also demonstrate the binding of the selective estrogen receptor modulators (SERMs) Raloxifene, Bazedoxifene, and a de novo derivative to the priming loop.Communicated by Ramaswamy H. Sarma.

The fraction of RNA that folds into the correct branched secondary structure determines hepatitis delta virus type 3 RNA editing levels.

RNA editing by the host RNA adenosine deaminase ADAR1 at the amber/W site of hepatitis delta virus RNA plays a central role in the viral replication cycle by affecting the balance between viral RNA synthesis and packaging. Previously, we found that HDV genotype III (HDV-3) RNA can form two secondary structures following transcription: an unbranched rod structure, which is characteristic of HDV, and a metastable branched structure that serves as the substrate for editing. The unstable nature of the branched editing substrate structure raised the possibility that structural dynamics of the RNA following transcription could determine the rate at which editing occurs. Here, editing and its control are examined in two HDV-3 isolates, from Peru and Ecuador. Analysis of editing in vitro by ADAR1 indicated that the branched structure formed by RNA derived from the Peruvian isolate is edited more efficiently than that from the Ecuadorian isolate. In contrast, in the context of replication, Peruvian RNA is edited less efficiently than RNA containing Ecuadorian sequences. Computational analyses of RNA folding using the massively parallel genetic algorithm (MPGAfold) indicated that the Peruvian RNA is less likely to form the branched structure required for editing than the Ecuadorian isolate. This difference was confirmed by in vitro transcription of these RNAs. Overall, our data indicate that HDV-3 controls RNA editing levels via (1) the fraction of the RNA that folds, during transcription, into the metastable branched structure required for editing and (2) the efficiency with which ADAR1 edits this branched substrate RNA.

Truncated tetrahedral RNA nanostructures exhibit enhanced features for delivery of RNAi substrates.

Using RNA as a material for nanoparticle construction provides control over particle size and shape at the nano-scale. RNA nano-architectures have shown promise as delivery vehicles for RNA interference (RNAi) substrates, allowing multiple functional entities to be combined on a single particle in a programmable fashion. Rather than employing a completely bottom-up approach to scaffold design, here multiple copies of an existing synthetic supramolecular RNA nano-architecture serve as building blocks along with additional motifs for the design of a novel truncated tetrahedral RNA scaffold, demonstrating that rationally designed RNA assemblies can themselves serve as modular pieces in the construction of larger rationally designed structures. The resulting tetrahedral scaffold displays enhanced characteristics for RNAi-substrate delivery in comparison to similar RNA-based scaffolds, as evidenced by its increased functional capacity, increased cellular uptake and ultimately an increased RNAi efficacy of its adorned Dicer substrate siRNAs. The unique truncated tetrahedral shape of the nanoparticle core appears to contribute to this particle's enhanced function, indicating the physical characteristics of RNA scaffolds merit significant consideration when designing platforms for delivery of functional RNAs via RNA nanoparticles.