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.

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.

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 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.

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.

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.

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.

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.

Structural Differences between Pri-miRNA Paralogs Promote Alternative Drosha Cleavage and Expand Target Repertoires.

MicroRNA (miRNA) processing begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the maturation of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its distorted and flexible stem structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Analyses of low-grade glioma patient samples indicate that the alternative-miR-9 has a potential role in tumor progression. Furthermore, we provide evidence that distortion of pri-miRNA stems induced by asymmetric internal loops correlates with Drosha cleavage at non-canonical sites. Our studies reveal that pri-miRNA paralogs can have distinct functions via differential Drosha processing.

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.

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.

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.

Protocols for Molecular Dynamics Simulations of RNA Nanostructures.

Molecular dynamics (MD) simulations have been used as one of the main research tools to study a wide range of biological systems and bridge the gap between X-ray crystallography or NMR structures and biological mechanism. In the field of RNA nanostructures, MD simulations have been used to fix steric clashes in computationally designed RNA nanostructures, characterize the dynamics, and investigate the interaction between RNA and other biomolecules such as delivery agents and membranes.In this chapter we present examples of computational protocols for molecular dynamics simulations in explicit and implicit solvent using the Amber Molecular Dynamics Package. We also show examples of post-simulation analysis steps and briefly mention selected tools beyond the Amber package. Limitations of the methods, tools, and protocols are also discussed. Most of the examples are illustrated for a small RNA duplex (helix), but the protocols are applicable to any nucleic acid structure, subject only to the computational speed and memory limitations of the hardware available to the user.

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.

Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch.

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.

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.

Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing.

Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.