Rotavirus RNA chaperone mediates global transcriptome-wide increase in RNA backbone flexibility

Due to genome segmentation, rotaviruses must co-package a set of eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus (RV) NSP2 protein. While the activities of distinct viral RNA chaperones are well studied on synthetic RNA substrates, little is known about their global effect on the entire viral transcriptome. Here we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to systematically examine the secondary structure of the RV transcriptome composed of eleven distinct transcripts in the absence and presence of increasing concentrations of RV NSP2. Surprisingly, SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate RV transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of the per nucleotide mutation rate measured by mutational profiling, from which SHAPE reactivities are derived, reveals a global five-fold rate increase in the presence of molar excess of NSP2. We demonstrate that the standard normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone activity. Further analysis of the mutation rate in the context of structural classification reveals a larger effect on stems rather than loop elements. Together, these data provide the first experimentally derived secondary structure model of the RV transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner, consistent with its promiscuous RNA-binding nature.

140 Allogeneic CAR T cells with deoxycytidine kinase knockdown demonstrate resistance to fludarabine

BackgroundClinical outcomes in CAR T therapy correlate with engraftment, expansion, and persistence of CAR T cells. In order to facilitate engraftment and expansion, a lymphodepleting regimen consisting of cyclophosphamide and fludarabine precedes CAR T infusion. This creates niches for infused CAR T cells and stimulates beneficial homeostatic cytokine production. As these compounds are also toxic to CAR T cells, administering the proper doses of both the conditioning drugs and the cell therapies with appropriate timing can be a challenge.MethodsTo protect CAR T cells from fludarabine toxicity, we have knocked down deoxycytidine kinase (dCK), which converts fludarabine from the prodrug form to an active compound. This was accomplished using an RNAi sequence featuring a dCK-specific shRNA sequence embedded into a micro-RNA backbone. The resulting RNAi sequence demonstrated the potency of shRNA and the stability of a microRNA. Using Precision BioSciences’ ARCUS® gene editing technology and AAV-mediated targeted transgene insertion strategy, we disrupted the endogenous T cell receptor and inserted a transgene encoding a CD19-specific CAR and a dCK-specific RNAi sequence. Cells produced in this manner were exposed to CD19+ target cells in vitro and in immune-deficient mice and CAR T proliferation and target killing were monitored in the presence and absence of fludarabine.ResultsWe observed that the inclusion of the RNAi feature resulted in 70% reduction in dCK mRNA abundance, and conferred resistance to fludarabine in vitro. Moreover, treatment of tumor-bearing mice with fludarabine and dCK knockdown CAR T cells resulted in enhanced tumor clearance and survival compared to mice receiving CAR T cells alone or fludarabine plus dCK replete CAR T cells.ConclusionsCAR T cells expressing a dCK-specific RNAi gene exhibited resistance to fludarabine in vitro and in vivo. This drug resistance feature may enable allogeneic CAR-T cells to be simultaneously administered with fludarabine, suppressing rejection of CAR T and improving CAR T engraftment and expansion. This synergy between conditioning and CAR T therapy may improve clinical outcomes by enhancing effector persistence and tumor clearing.AcknowledgementsI would like to thank Aaron J. Martin, PhD and Daniel T. Macleod, PhD for their excellent mentorship and the Precision Biosciences Vivarium team for their support during this study.ReferencesMacleod DT, et al. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells. Mol Ther 2017; 25(4):949–961.Fellmann C, et al. An Optimized microRNA Backbone for Effective Single-Copy RNAi. Cell Reports 2013;5:1704–1713.Ethics ApprovalThe animal study conducted was approved by the Institutional Animal Care and Use Committee (IACUC) of Mispro Biotech.

Physicochemical and Toxicity Investigation of Chitosan-based dsRNA Nanocarrier Formation

Technologies involving the use of double-stranded RNA (dsRNA) to elicit RNA interference (RNAi) in pest control have emerged as an alternative to traditional pesticides. RNAi can mediate natural cell protection being a promising tool to provide prompt responses in plant defense against pathogens. The present study is focused on the physicochemical characterization of formed dsRNA-loaded nanoparticles as a result of chitosan-dsRNA ionic interactions. Additionally, a preliminary investigation was conducted of the in-vitro toxicity of loaded nanoparticles in lettuce and human red blood cells. dsRNA molecules, homologous to partial phytopathogenic tomato mosaic virus (ToMV) sequence, were used as a model. The main groups involved in the chitosan-dsRNA ionic coupling were identified by Fourier-transform infrared spectroscopy, and the stability of formed nanoparticles was accessed by dynamic light scattering, electrophoresis, and thermal analyses. The chitosan showed a higher ability to bind to dsRNA at low charge ratios (N/P = 1), ruled by positively charged chitosan methyl groups and negatively charged phosphate groups from the RNA backbone, resulting in small nanoparticles (73.25 nm size) at low polydispersity (0.25). The toxic assays of these particles, on lettuce seeds and in human erythrocytes, revealed very low toxicity demonstrating their safety as a platform, thereby holding potential use as biodefensive for crop protection.

Non-enzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides

Aminoacylated tRNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA World prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA World. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid-RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis.

Learning torus PCA based classification for multiscale RNA backbone structure correction with application to SARS-CoV-2

Reconstructions of structure of biomolecules, for instance via X-ray crystallography or cryo-EM frequently contain clashes of atomic centers. Correction methods are usually based on simulations approximating biophysical chemistry, making them computationally expensive and often not correcting all clashes. We propose a computationally fast data-driven statistical method yielding suites free from within-suite clashes: From such a clash free training data set, devising mode hunting after torus PCA on adaptive cutting average linkage tree clustering (MINTAGE), we learn RNA suite shapes. With classification based on multiscale structure enhancement (CLEAN), for a given clash suite we determine its neighborhood on a mesoscopic scale involving several suites. As corrected suite we propose the Fréchet mean on a torus of the largest classes in this neighborhood. We validate CLEAN MINTAGE on a benchmark data set, compare it to a state of the art correction method and apply it, as proof of concept, to two exemplary suites adjacent to helical pieces of the frameshift stimulation element of SARS-CoV-2 which are difficult to reconstruct. In contrast to a recent reconstruction proposing several different structure models, CLEAN MINTAGE unanimously proposes structure corrections within the same clash free class for all suites.

Pairing a high-resolution statistical potential with a nucleobase-centric sampling algorithm for improving RNA model refinement

Refining modelled structures to approach experimental accuracy is one of the most challenging problems in molecular biology. Despite many years’ efforts, the progress in protein or RNA structure refinement has been slow because the global minimum given by the energy scores is not at the experimentally determined “native” structure. Here, we propose a fully knowledge-based energy function that captures the full orientation dependence of base–base, base–oxygen and oxygen–oxygen interactions with the RNA backbone modelled by rotameric states and internal energies. A total of 4000 quantum-mechanical calculations were performed to reweight base–base statistical potentials for minimizing possible effects of indirect interactions. The resulting BRiQ knowledge-based potential, equipped with a nucleobase-centric sampling algorithm, provides a robust improvement in refining near-native RNA models generated by a wide variety of modelling techniques.

Phosphorothioate Substitutions in RNA Structure Studied by Molecular Dynamics Simulations, QM/MM Calculations and NMR Experiments

Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single non-bridging oxygen of the phosphate is replaced with a sulphur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as tool to explore their function. It is, however, unclear what basic physical effects PT has on RNA stability and electronic structure. Here, we present Molecular Dynamics (MD) simulations, quantum mechanical (QM) calculations, and NMR spectroscopy measurements, exploring the effects of PT modifications in the structural context of the Neomycin-sensing riboswitch (NSR). The NSR is the smallest biologically functional riboswitch with a well-defined structure stabilized by a U-turn motif. Three of the signature interactions of the U-turn; an H-bond, an anion-π interaction and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA structure and dynamics. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulphur substitution, besides weakening the direct H-bond interaction, reduces the directionality of H-bonding while increasing its dispersion and induction components. It also reduces the induction and increases dispersion component of the anion-π stacking. The sulphur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to unsatisfactory description of PT in simulations of the NSR. We show that it is not possible to accurately describe the PT interactions using one universal set of van der Waals sulphur parameters and provide suggestions for improving the force-field performance.

Single amino acid-promoted reactions link a non-enzymatic chemical network to the early evolution of enzymatic pentose phosphate pathway

How metabolic pathways emerged in early evolution remains largely unknown. Recently discovered chemical networks driven by iron and sulfur resemble reaction sequences found within glycolysis, gluconeogenesis, the oxidative and reductive Krebs cycle, the Wood Ljungdahl as well as the S-adenosylmethionine pathways, components of the core cellular metabolic network. These findings suggest that the evolution of central metabolism was primed by environmental chemical reactions, implying that non-enzymatic reaction networks served as a “template” in the evolution of enzymatic activities. We speculated that the turning point for this transition would depend on the catalytic properties of the simplest structural components of proteins, single amino acids. Here, we systematically combine constituents of Fe(II)-driven non-enzymatic reactions resembling glycolysis and pentose phosphate pathway (PPP), with single proteinogenic amino acids. Multiple reaction rates are enhanced by amino acids. In particular, cysteine is able to replace (and/or complement) the metal ion Fe(II) in driving the non-enzymatic formation of the RNA-backbone metabolite ribose 5-phosphate from 6-phosphogluconate, a rate-limiting reaction of the oxidative PPP. In the presence of both Fe(II) and cysteine, a complex is formed, enabling the non-enzymatic reaction to proceed at a wide range of temperatures. At mundane temperatures, this ‘minimal enzyme-like complex’ achieves a much higher specificity in the formation of ribose 5-phosphate than the Fe(II)-driven reaction at high temperatures. Hence, simple amino acids can accelerate key steps within metal-promoted metabolism-like chemical networks. Our results imply a stepwise scenario, in which environmental chemical networks served as primers in the early evolution of the metabolic network structure.Significance StatementThe evolutionary roots of metabolic pathways are barely understood. Here we show results consistent with a stepwise scenario during the evolution of (enzymatic) metabolism, starting from non-enzymatic chemical networks. By systematic screening of metabolic-like reactivities in vitro, and using high-throughput analytical techniques, we identify an iron/cysteine complex to act as a ‘minimal enzymelike complex’, which consists of a metal ion, an amino acid, and a sugar phosphate ligand. Integrated in a metal-driven, non-enzymatic pentose phosphate pathway, it promotes the formation of the RNA-backbone precursor ribose 5-phosphate at ambient temperature.