Predicting RNA SHAPE scores with deep learning.

Secondary structure prediction approaches rely typically on models of equilibrium free energies that are themselves based on in vitro physical chemistry. Recent transcriptome-wide experiments of in vivo RNA structure based on SHAPE-MaP experiments provide important information that may make it possible to extend current in vitro-based RNA folding models in order to improve the accuracy of computational RNA folding simulations with respect to the experimentally measured in vivo RNA secondary structure. Here we present a machine learning approach that utilizes RNA secondary structure prediction results and nucleotide sequence in order to predict in vivo SHAPE scores. We show that this approach has a higher Pearson correlation coefficient with experimental SHAPE scores than thermodynamic folding. This could be an important step towards augmenting experimental results with computational predictions and help with RNA secondary structure predictions that inherently take in-vivo folding properties into account.

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.

A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides.

Several varieties of small nucleic acid constructs are able to modulate gene expression via one of a number of different pathways and mechanisms. These constructs can be synthesized, assembled and delivered to cells where they are able to impart regulatory functions, presenting a potential avenue for the development of nucleic acid-based therapeutics. However, distinguishing aberrant cells in need of therapeutic treatment and limiting the activity of deliverable nucleic acid constructs to these specific cells remains a challenge. Here, we designed and characterized a collection of nucleic acids systems able to generate and/or release sequence-specific oligonucleotide constructs in a conditional manner based on the presence or absence of specific RNA trigger molecules. The conditional function of these systems utilizes the implementation of AND and NOT Boolean logic elements, which could ultimately be used to restrict the release of functionally relevant nucleic acid constructs to specific cellular environments defined by the high or low expression of particular RNA biomarkers. Each system is generalizable and designed with future therapeutic development in mind. Every construct assembles through nuclease-resistant RNA/DNA hybrid duplex formation, removing the need for additional 2'-modifications, while none contain any sequence restrictions on what can define the diagnostic trigger sequence or the functional oligonucleotide output.

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.

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.

Computational Generation of RNA Nanorings.

A variety of designed RNA ring structures (ranging from triangles to hexagonal rings) have been reported in the scientific literature. Designing self-assembling RNA ring structures from structural motifs is, however, a nontrivial problem as there are many combinations of motifs and linking helices. Moreover, most combinations of motifs and linker helices will not lead to ring closure. A solution to this problem was recently published using a "design-by-catalog" approach where motif combinations that lead to rings are precomputed and tabulated. Here we present a web-browser based workflow for creating RNA rings using Galaxy, a web-based platform that can be used for workflow management. An example of how these RNA rings are generated and processed to create a 3D model of the ring is discussed.

Preparation of a Conditional RNA Switch.

RNA has gained great interest for use in biomedical and therapeutic applications. This is due in part to RNA's ability to perform multiple functions, including the regulation of endogenously expressed genes. However, the ability of RNA based drugs to distinguish target diseased cells from healthy tissue remains challenging. Here we present methods for the production of a recently developed conditional RNA switch that releases a Dicer substrate RNA in response to interaction with a specific RNA biomarker.

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.

Triggerable RNA nanodevices.

The targeted and conditional activation of pharmaceuticals is an increasingly important feature in modern personalized medicine. Nucleic acid nanoparticles show tremendous potential in this exploit due to their programmability and biocompatibility. Among the most powerful nucleic acid specific treatments is RNA interference-based therapeutics. RNA interference is a naturally occurring phenomenon in which specific genes are effectively silenced. Recently we have developed two different strategies based on customized multivalent nucleic acid nanoparticles with the ability to conditionally activate RNA interference in diseased cells as well as elicit detectable fluorescent responses.[1,2] These novel technologies can be further utilized for the simultaneous delivery and conditional intracellular activation of multiple therapeutic and biosensing functions to combat various diseases.

Cellular Delivery of RNA Nanoparticles.

RNA nanostructures can be programmed to exhibit defined sizes, shapes and stoichiometries from naturally occurring or de novo designed RNA motifs. These constructs can be used as scaffolds to attach functional moieties, such as ligand binding motifs or gene expression regulators, for nanobiology applications. This review is focused on four areas of importance to RNA nanotechnology: the types of RNAs of particular interest for nanobiology, the assembly of RNA nanoconstructs, the challenges of cellular delivery of RNAs in vivo, and the delivery carriers that aid in the matter. The available strategies for the design of nucleic acid nanostructures, as well as for formulation of their carriers, make RNA nanotechnology an important tool in both basic research and applied biomedical science.

Ring Catalog: A resource for designing self-assembling RNA nanostructures.

Designing self-assembling RNA ring structures based on known 3D structural elements connected via linker helices is a challenging task due to the immense number of motif combinations, many of which do not lead to ring-closure. We describe an in silico solution to this design problem by combinatorial assembly of RNA 3-way junctions, bulges, and kissing loops, and tabulating the cases that lead to ring formation. The solutions found are made available in the form of a web-accessible Ring Catalog. As an example of a potential use of this resource, we chose a predicted RNA square structure consisting of five RNA strands and demonstrate experimentally that the self-assembly of those five strands leads to the formation of a square-like complex. This is a demonstration of a novel "design by catalog" approach to RNA nano-structure generation. The URL https://rnajunction.ncifcrf.gov/ringdb can be used to access the resource.

The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities.

Current work reports the use of single-stranded RNA toeholds of different lengths to promote the reassociation of various RNA-DNA hybrids, which results in activation of multiple split functionalities inside human cells. The process of reassociation is analyzed and followed with a novel computational multistrand secondary structure prediction algorithm and various experiments. All of our previously designed RNA/DNA nanoparticles employed single-stranded DNA toeholds to initiate reassociation. The use of RNA toeholds is advantageous because of the simpler design rules, the shorter toeholds, and the smaller size of the resulting nanoparticles (by up to 120 nucleotides per particle) compared to the same hybrid nanoparticles with single-stranded DNA toeholds. Moreover, the cotranscriptional assemblies result in higher yields for hybrid nanoparticles with ssRNA toeholds.

Multistrand Structure Prediction of Nucleic Acid Assemblies and Design of RNA Switches.

RNA is an attractive material for the creation of molecular logic gates that release programmed functionalities only in the presence of specific molecular interaction partners. Here we present HyperFold, a multistrand RNA/DNA structure prediction approach for predicting nucleic acid complexes that can contain pseudoknots. We show that HyperFold also performs competitively compared to other published folding algorithms. We performed a large variety of RNA/DNA hybrid reassociation experiments for different concentrations, DNA toehold lengths, and G+C content and find that the observed tendencies for reassociation correspond well to computational predictions. Importantly, we apply this method to the design and experimental verification of a two-stranded RNA molecular switch that upon binding to a single-stranded RNA toehold disease-marker trigger mRNA changes its conformation releasing an shRNA-like Dicer substrate structure. To demonstrate the concept, connective tissue growth factor (CTGF) mRNA and enhanced green fluorescent protein (eGFP) mRNA were chosen as trigger and target sequences, respectively. In vitro experiments confirm the formation of an RNA switch and demonstrate that the functional unit is being released when the trigger RNA interacts with the switch toehold. The designed RNA switch is shown to be functional in MDA-MB-231 breast cancer cells. Several other switches were also designed and tested. We conclude that this approach has considerable potential because, in principle, it allows the release of an siRNA designed against a gene that differs from the gene that is utilized as a biomarker for a disease state.

Computational and experimental studies of reassociating RNA/DNA hybrids containing split functionalities.

Recently, we developed a novel technique based on RNA/DNA hybrid reassociation that allows conditional activation of different split functionalities inside diseased cells and in vivo. We further expanded this idea to permit simultaneous activation of multiple different functions in a fully controllable fashion. In this chapter, we discuss some novel computational approaches and experimental techniques aimed at the characterization, design, and production of reassociating RNA/DNA hybrids containing split functionalities. We also briefly describe several experimental techniques that can be used to test these hybrids in vitro and in vivo.

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.

Genomic analyses reveal broad impact of miR-137 on genes associated with malignant transformation and neuronal differentiation in glioblastoma cells.

miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137--among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFß2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.

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.

Computational and experimental characterization of RNA cubic nanoscaffolds.

The fast-developing field of RNA nanotechnology requires the adoption and development of novel and faster computational approaches to modeling and characterization of RNA-based nano-objects. We report the first application of Elastic Network Modeling (ENM), a structure-based dynamics model, to RNA nanotechnology. With the use of an Anisotropic Network Model (ANM), a type of ENM, we characterize the dynamic behavior of non-compact, multi-stranded RNA-based nanocubes that can be used as nano-scale scaffolds carrying different functionalities. Modeling the nanocubes with our tool NanoTiler and exploring the dynamic characteristics of the models with ANM suggested relatively minor but important structural modifications that enhanced the assembly properties and thermodynamic stabilities. In silico and in vitro, we compared nanocubes having different numbers of base pairs per side, showing with both methods that the 10 bp-long helix design leads to more efficient assembly, as predicted computationally. We also explored the impact of different numbers of single-stranded nucleotide stretches at each of the cube corners and showed that cube flexibility simulations help explain the differences in the experimental assembly yields, as well as the measured nanomolecule sizes and melting temperatures. This original work paves the way for detailed computational analysis of the dynamic behavior of artificially designed multi-stranded RNA nanoparticles.

The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus-induced microRNAs.

IFNL3, which encodes interferon-λ3 (IFN-λ3), has received considerable attention in the hepatitis C virus (HCV) field, as many independent genome-wide association studies have identified a strong association between polymorphisms near IFNL3 and clearance of HCV. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) in the 3' untranslated region (UTR) of IFNL3 mRNA that dictated transcript stability. We found that this polymorphism influenced AU-rich element (ARE)-mediated decay (AMD) of IFNL3 mRNA, as well as the binding of HCV-induced microRNAs during infection. Together these pathways mediated robust repression of the unfavorable IFNL3 polymorphism. Our data reveal a previously unknown mechanism by which HCV attenuates the antiviral response and indicate new potential therapeutic targets for HCV treatment.