ABSTRACT
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.
Subject(s)
Hepatitis B virus , Hepatitis B , Humans , Virus Replication , RNA, Viral/genetics , GenomicsABSTRACT
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.
Subject(s)
Molecular Dynamics Simulation , Proteins , Binding Sites , Ligands , Molecular Conformation , Molecular Docking Simulation , Protein Binding , Protein Conformation , Proteins/chemistry , RNA/metabolismABSTRACT
We previously reported that hydroxylated oxime ether lipids (OELs) efficiently deliver functional Dicer substrate siRNAs (DsiRNAs) in cells. Here, we explored in vivo utility of these OELs, using OEL4 as a prototype and report that surface modification of the OEL4 formulations was essential for their in vivo applications. These surface-modified OEL4 formulations were developed by inclusion of various PEGylated lipids. The vesicle stability and gene knock-down were dependent on the PEG chain length. OEL4 containing DSPE-PEG350 and DSPE-PEG1000 (surprisingly not DSPE2000) promoted gene silencing in cells. In vivo studies demonstrated that OEL4 vesicles formulated using 3 mol% DSPE-PEG350 accumulate in human lung cancer (A549-luc2) xenografts in mice and exhibit a significant increase in tumor to liver ratios. These vesicles also showed a statistically significant reduction of luciferase signal in tumors compared to untreated mice. Taken together, the scalable OEL4:DSPE-PEG350 formulation serves as a novel candidate for delivery of RNAi therapeutics.
Subject(s)
Ether , Lung Neoplasms , Animals , Ethers , Heterografts , Humans , Lipids , Lung Neoplasms/genetics , Lung Neoplasms/therapy , Mice , Oximes , Polyethylene Glycols , RNA, Small Interfering/geneticsABSTRACT
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.
Subject(s)
Hepatitis B virus , Nucleic Acid Conformation , RNA, Viral , Virus Replication , Genomics , Hepatitis B virus/genetics , Hepatitis B virus/physiology , Humans , RNA, Viral/chemistry , Reverse TranscriptionABSTRACT
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.
Subject(s)
Protein Biosynthesis , Tombusviridae , 3' Untranslated Regions , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Nucleic Acid Conformation , RNA, Viral/chemistry , Tombusviridae/physiologyABSTRACT
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.
Subject(s)
Hepatitis B virus , RNA, Viral , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , RNA, Viral/chemistry , Genomics , Virus ReplicationABSTRACT
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.
Subject(s)
Herpesvirus 8, Human/metabolism , Nucleotides/metabolism , Poly A/metabolism , RNA, Long Noncoding/metabolism , RNA, Viral/metabolism , Small Molecule Libraries/metabolism , Base Sequence , Crystallography, X-Ray , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/physiology , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Nucleic Acid Conformation , Nucleotides/genetics , Poly A/chemistry , Poly A/genetics , RNA Stability/genetics , RNA, Long Noncoding/chemistry , RNA, Long Noncoding/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Sarcoma, Kaposi/virology , Small Molecule Libraries/chemistryABSTRACT
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.
Subject(s)
Computational Biology , Deep Learning , Models, Molecular , Nucleic Acid Conformation , RNA Folding , RNA/chemistry , Codon, Initiator , Computational Biology/methods , Neural Networks, Computer , RNA/geneticsABSTRACT
Small interfering RNAs (siRNAs) are potential therapeutic substances due to their gene silencing capability as exemplified by the recent approval by the US Food and Drug Administration (FDA) of the first siRNA therapeutic agent (patisiran). However, the delivery of naked siRNAs is challenging because of their short plasma half-lives and poor cell penetrability. In this study, we used vesicles made from bolaamphiphiles (bolas), GLH-19 and GLH-20, to investigate their ability to protect siRNA from degradation by nucleases while delivering it to target cells, including cells in the brain. Based on computational and experimental studies, we found that GLH-19 vesicles have better delivery characteristics than do GLH-20 vesicles in terms of stability, binding affinity, protection against nucleases, and transfection efficiency, while GLH-20 vesicles contribute to efficient release of the delivered siRNAs, which become available for silencing. Our studies with vesicles made from a mixture of the two bolas (GLH-19 and GLH-20) show that they were able to deliver siRNAs into cultured cancer cells, into a flank tumor and into the brain. The vesicles penetrate cell membranes and the blood-brain barrier (BBB) by endocytosis and transcytosis, respectively, mainly through the caveolae-dependent pathway. These results suggest that GLH-19 strengthens vesicle stability, provides protection against nucleases, and enhances transfection efficiency, while GLH-20 makes the siRNA available for gene silencing.
ABSTRACT
Translation potential of RNA interference nanotherapeutics remains challenging due to in vivo off-target effects and poor endosomal escape. Here, we developed novel polyplexes for controlled intracellular delivery of dicer substrate siRNA, using a light activation approach. Sulfonated polyethylenimines covalently linked to pyropheophorbide-α for photoactivation and bearing modified amines (sulfo-pyro-PEI) for regulated endosomal escape were investigated. Gene knock-down by the polymer-complexed DsiRNA duplexes (siRNA-NPs) was monitored in breast cancer cells. Surprisingly, sulfo-pyro-PEI/siRNA-NPs failed to downregulate the PLK1 or eGFP proteins. However, photoactivation of these cell associated-polyplexes with a 661-nm laser clearly restored knock-down of both proteins. In contrast, protein down-regulation by non-sulfonated pyro-PEI/siRNA-NPs occurred without any laser treatments, indicating cytoplasmic disposition of DsiRNA followed a common intracellular release mechanism. Therefore, sulfonated pyro-PEI holds potential as a unique trap and release light-controlled delivery platform for on-demand gene silencing bearing minimal off target effects.
Subject(s)
Breast Neoplasms/genetics , Cell Cycle Proteins/genetics , DEAD-box RNA Helicases/genetics , Gene Silencing , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Ribonuclease III/genetics , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cell Line, Tumor , Endosomes/drug effects , Female , Gene Knockdown Techniques , Green Fluorescent Proteins/genetics , Humans , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , Polymers/chemistry , RNA Interference , RNA, Small Interfering/pharmacology , Polo-Like Kinase 1ABSTRACT
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.
Subject(s)
DEAD-box RNA Helicases/chemistry , Nanostructures/chemistry , RNA Interference , RNA/chemistry , Ribonuclease III/chemistry , Cell Cycle Proteins/chemistry , Cell Line, Tumor , Cryoelectron Microscopy , Green Fluorescent Proteins/chemistry , Humans , Light , Molecular Dynamics Simulation , Nucleic Acid Conformation , Particle Size , Polymerase Chain Reaction , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , RNA, Small Interfering , Scattering, Radiation , Software , Thermodynamics , Polo-Like Kinase 1ABSTRACT
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.
Subject(s)
Algorithms , RNA/chemistry , Software , Animals , Base Pairing , Base Sequence , Computer Graphics , Humans , Information Storage and Retrieval , Nucleic Acid Conformation , RNA/genetics , RNA/metabolism , Viruses/genetics , Viruses/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , Ribonuclease III/metabolism , Brain Neoplasms/genetics , Glioma/genetics , HEK293 Cells , HeLa Cells , Humans , MicroRNAs/chemistry , MicroRNAs/geneticsABSTRACT
A second-generation chlorin-based photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) has shown tremendous therapeutic potential in clinical trials in the treatment of esophageal cancer. Herein, we have developed and validated a bioanalytical method for estimation of HPPH in rat plasma using High Performance Liquid Chromatography (HPLC) with a photo diode array (PDA) detector. The method was applied for carrying out pharmacokinetic study of HPPH. Further pharmacokinetic modeling was carried out to understand the compartment kinetics of HPPH. The developed method was fully validated as per the United States Food and Drug Administration (US-FDA) guidelines for bioanalytical method validation. The linearity of the method was in the range of 250-8000 ng mL-1, and the plasma recovery was found to be 70%. Pharmacokinetic parameters were evaluated and compared via non-compartment analysis and compartment modeling after the intravenous (i.v.) bolus administration in rats using Phoenix WinNonlin 8.0 (Certara™, USA). From the obtained results, we hypothesize that the HPPH complies with two compartmental pharmacokinetic model. Furthermore, it was observed that HPPH has the rapid distribution from the central compartment to peripheral compartment along with slow elimination from peripheral compartment.
Subject(s)
Chlorophyll/analogs & derivatives , Photosensitizing Agents/pharmacokinetics , Animals , Chlorophyll/administration & dosage , Chlorophyll/blood , Chlorophyll/pharmacokinetics , Chromatography, High Pressure Liquid , Injections, Intravenous , Kinetics , Photosensitizing Agents/administration & dosage , Photosensitizing Agents/blood , Rats , Rats, WistarABSTRACT
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.
Subject(s)
RNA, Double-Stranded/metabolism , RNA, Double-Stranded/therapeutic use , RNA-Binding Proteins/metabolism , Base Sequence , Endoribonucleases/metabolism , HEK293 Cells , Humans , Protein Binding , RNA, Double-Stranded/chemistry , RNA, Ribosomal/metabolism , Ribosomes/metabolism , SolventsABSTRACT
Advances in in vivo stability and preferential tumor uptake of cancer nanomedicine are warranted for effective chemotherapy. Here, we describe a novel nanoformulation using an unconventional polymeric tubule-forming phospholipid, DC8,9PC. We report that DC8,9PC transitions to stable vesicles (LNPs) in the presence of PEGylated lipid (DSPE-PEG2000); the resulting DC8,9PC:DSPE-PEG2000 LNPs efficiently included a hydrophobic PDT drug, HPPH. Remarkably, these LNPs incorporated unusually high DSPE-PEG2000 concentrations; LNP10-HPPH and LNP20-HPPH (10 & 20 mol% PEGylated lipid, respectively) exhibited >90% serum stability at 37⯰C. Increased PEGylation in the LNPs correlated with enhanced tumor accumulation in intravenously injected HT29 tumor mouse xenographs. Colon-26 bearing BALB/c mice, intravenously injected with LNP20-HPPH showed superior PDT efficacy and animal survival (no tumor recurrence up to 100 days) as compared to a formulation currently used in clinical trials. Taken together, we present a simple stealth binary lipid nanosystem with enhanced efficiency of tumor accumulation and superior therapeutic efficacy.
Subject(s)
Colorectal Neoplasms/drug therapy , Drug Delivery Systems , Nanoparticles/administration & dosage , Phospholipids/chemistry , Photochemotherapy , Photosensitizing Agents/administration & dosage , Polymers/chemistry , Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Colorectal Neoplasms/pathology , Drug Carriers/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Mice , Mice, Inbred BALB C , Mice, Nude , Nanoparticles/chemistry , Photosensitizing Agents/chemistry , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , RNA/chemistry , Software , Algorithms , Computer GraphicsABSTRACT
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.
Subject(s)
Air , Aluminum Silicates/chemistry , Nanoparticles/chemistry , RNA/chemistry , Cell Line, Tumor , Humans , Leukocytes, Mononuclear/immunology , Microscopy, Atomic Force/methods , Molecular Dynamics Simulation , Nucleic Acid Conformation , Pliability , RNA/immunologyABSTRACT
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.