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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Structure of 3'-PO 4 /5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)–GMP, RNA 3'-phosphate, and RNA 3 'pp 5 'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. In this work, we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide HO A 1 pT 2 pG 3 pT 4 pC 5 pC 6 , which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the A1pT2 and T2pG3 phosphates; Arg190 and Gln194 to the T 2 pG 3 phosphate; and an Arg190 π-cation interaction with the G 3 nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Modification at the C2'-O-Position with 2-Methylbenzothiophene Induces Unique Structural Changes and Thermal Transitions on Duplexes of RNA and DNA

Oligonucleotides can be chemically modified for a variety of applications that include their use as biomaterials, in therapeutics, or as tools to understand biochemical processes, among others. This work focuses on the functionalization of oligonucleotides of RNA and DNA (12- or 14-nucleotides long) with methylbenzothiophene (BT), at the C2'-O-position, which led to unique structural features. Circular dichroism (CD) analyses showed that positioning the BT units on one strand led to significant thermal destabilization, while duplexes where each strand contained 4-BT rings formed a distinct arrangement with cooperativity/interactions among the modifications (evidenced from the appearance of a band with positive ellipticity at 235 nm). Interestingly, the structural arrays displayed increased duplex stabilization (>10 °C higher than the canonical analogue) as a function of [Na + ] with an unexpected structural rearrangement at temperatures above 50 °C. Density functional theory–polarizable continuum model (DFT-PCM) calculations were carried out, and the analyses were in agreement with induced structural changes as a function of salt content. A model was proposed where the hydrophobic surface allows for an internal nucleobase rearrangement into a more thermodynamically stable structure, before undergoing full denaturation, with increased heat. While this behavior is not common, B- to Z-form duplex transitions can occur and are dependent on parameters that were probed in this work, i.e., temperature, nature of modification, or ionic content. To take advantage of this phenomenon, we probed the ability of the modified duplexes to be recognized by Zα (an RNA binding protein that targets Z-form RNA) via electrophoretic analysis and CD. Interestingly, the protein did not bind to canonical duplexes of DNA or RNA; however, it recognized the modified duplexes, in a [monovalent/divalent salt] dependent manner. Overall, the findings describe methodology to attain unique structural motifs of modified duplexes of DNA or RNA, and control their behavior as a function of salt concentration. While their affinity to RNA binding proteins, and the corresponding mechanism of action, requires further exploration, the tunable properties can be of potential use to study this, and other, types of modifications. The novel arrays that formed, under the conditions described herein, provide a useful way to explore the structure and behavior of modified oligonucleotides, in general.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Visualizing a viral genome with contrast variation small angle X-ray scattering

Despite the threat to human health posed by some single-stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast variation small-angle X-ray scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, though present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detected with CV-SAXS and compared with a structure derived from asymmetric cryo-EM reconstructions. Additional comparisons with free RNA highlight the significant structural rearrangements induced by capsid proteins and invite the application of time-resolved CV-SAXS to reveal interactions that result in efficient viral assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Deep structural clustering for single-cell RNA-seq data jointly through autoencoder and graph neural network

Abstract Single-cell RNA sequencing (scRNA-seq) permits researchers to study the complex mechanisms of cell heterogeneity and diversity. Unsupervised clustering is of central importance for the analysis of the scRNA-seq data, as it can be used to identify putative cell types. However, due to noise impacts, high dimensionality and pervasive dropout events, clustering analysis of scRNA-seq data remains a computational challenge. Here, we propose a new deep structural clustering method for scRNA-seq data, named scDSC, which integrate the structural information into deep clustering of single cells. The proposed scDSC consists of a Zero-Inflated Negative Binomial (ZINB) model-based autoencoder, a graph neural network (GNN) module and a mutual-supervised module. To learn the data representation from the sparse and zero-inflated scRNA-seq data, we add a ZINB model to the basic autoencoder. The GNN module is introduced to capture the structural information among cells. By joining the ZINB-based autoencoder with the GNN module, the model transfers the data representation learned by autoencoder to the corresponding GNN layer. Furthermore, we adopt a mutual supervised strategy to unify these two different deep neural architectures and to guide the clustering task. Extensive experimental results on six real scRNA-seq datasets demonstrate that scDSC outperforms state-of-the-art methods in terms of clustering accuracy and scalability. Our method scDSC is implemented in Python using the Pytorch machine-learning library, and it is freely available at https://github.com/DHUDBlab/scDSC.

Gan, Yanglan↗
DOE OSTI2024

Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease

Abstract RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the human tRNA ligase.

Science & Technology - Other Topics↗
DOE OSTI2023

Crystal structure of a highly conserved enteroviral 5' cloverleaf RNA replication element

The extreme 5'-end of the enterovirus RNA genome contains a conserved cloverleaf-like domain that recruits 3CD and PCBP proteins required for initiating genome replication. Here, we report the crystal structure at 1.9 Å resolution of this domain from the CVB3 genome in complex with an antibody chaperone. The RNA folds into an antiparallel H-type four-way junction comprising four subdomains with co-axially stacked sA-sD and sB-sC helices. Long-range interactions between a conserved A40 in the sC-loop and Py-Py helix within the sD subdomain organize near-parallel orientations of the sA-sB and sC-sD helices. Our NMR studies confirm that these long-range interactions occur in solution and without the chaperone. The phylogenetic analyses indicate that our crystal structure represents a conserved architecture of enteroviral cloverleaf-like domains, including the A40 and Py-Py interactions. The protein binding studies further suggest that the H-shape architecture provides a ready-made platform to recruit 3CD and PCBP2 for viral replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Human Coronavirus-229E Hijacks Key Host-Cell RNA-Processing Complexes for Replication

The recent rise in zoonotic coronavirus outbreaks underscores the urgency to understand virus-host interactions and develop potent antiviral therapeutics. Systems biology approaches, particularly proteomics have been invaluable in providing a global overview of such interactions. However, these conventional approaches rely on measuring protein abundance changes which don’t reflect functional shifts. In this study, we employed a high-throughput structural proteomics approach called limited proteolysis-based mass spectrometry (LiP-MS) to capture conformational changes, which we demonstrate are better proxies for functional alterations. We applied this tool to both immortalized and primary human lung cells following human coronavirus 229E (HCoV-229E) infection. We identified significant infection-induced structural changes within RNA processing complexes such as the spliceosome-C and NOP56-associated complex. These observations emphasize that HCoV-229E infection propagates a multi-pronged effort to obstruct the house keeping RNA processing functions in the host. Finally, we show that HCoV-229E replication can be attenuated by the targeted disruption of these complexes, indicating that the identified cellular factories are viable targets to prevent coronavirus infection.

coronavirus↗
DOE OSTI2020

RNAcentral 2021: secondary structure integration, improved sequence search and new member databases

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data resource for the RNA community. RNAcentral is freely available at https://rnacentral.org.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA–RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K D

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

RNA nanotechnology to build a dodecahedral genome of single-stranded RNA virus

The quest for artificial RNA viral complexes with authentic structure while being non-replicative is on its way for the development of viral vaccines. RNA viruses contain capsid proteins that interact with the genome during morphogenesis. The sequence and properties of the protein and genome determine the structure of the virus. For example, the Pariacoto virus ssRNA genome assembles into a dodecahedron. Virus-inspired nanotechnology has progressed remarkably due to the unique structural and functional properties of viruses, which can inspire the design of novel nanomaterials. RNA is a programmable biopolymer able to self-assemble sophisticated 3D structures with rich functionalities. RNA dodecahedrons mimicking the Pariacoto virus quasi-icosahedral genome structures were constructed from both native and 2'-F modified RNA oligos. The RNA dodecahedron easily self-assembled using the stable pRNA three-way junction of bacteriophage phi29 as building blocks. The RNA dodecahedron cage was further characterized by cryo-electron microscopy and atomic force microscopy, confirming the spontaneous and homogenous formation of the RNA cage. The reported RNA dodecahedron cage will likely provide further studies on the mechanisms of interaction of the capsid protein with the viral genome while providing a template for further construction of the viral RNA scaffold to add capsid proteins for the assembly of the viral nucleocapsid as a model. Understanding the self-assembly and RNA folding of this RNA cage may offer new insights into the 3D organization of viral RNA genomes. Finally, the reported RNA cage also has the potential to be explored as a novel virus-inspired nanocarrier.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The Importance of Solution Studies for the Structural Characterization of the Enterovirus 5’ Cloverleaf

Enteroviruses initiate genomic replication via a highly conserved mechanism that is controlled by an RNA platform, also known as the 5’ cloverleaf (5’CL). Here, we present a biophysical analysis of the 5’CL conformation of three enterovirus serotypes under various ionic conditions, utilizing CD spectroscopy, size-exclusion chromatography, and small-angle X-ray scattering. In general, a tendency toward a smaller monomeric hydrodynamic radius in the presence of salts was observed, but the exact structural signature of each 5’CL varied depending upon the serotype. Rhinovirus B14 (RVB14) exhibited at least two monomeric conformations and a low propensity for dimerization, while poliovirus 1 (PV1) showed a high propensity for dimerization, which was enhanced by the presence of salts. Enterovirus D70 was observed to be somewhat intermediate, with primarily a monomeric structure, but possessing some potential for dimerization. The equilibrium between the two monomeric and the dimeric conformations is also discussed. These results indicate that the 5’CL conformation may be more complex than the current literature suggests, thus underscoring the need for a combined crystal and solution approach for the accurate representation of the 5’CL conformation, and the conformation of other RNA structural elements, under native conditions.

Virology↗
DOE OSTI2025

Phenotypically anchored transcriptomics across diverse agrichemicals reveals conserved pathways and unique gene expression signatures in zebrafish

Agrichemicals such as herbicides, fungicides, insecticides, and biocides are widely used in agriculture, yet some are associated with adverse effects in humans and the environment. While many of these chemicals have been extensively studied in vitro and are included in the EPA’s ToxCast program, comprehensive in vivo comparisons using RNA sequencing across structurally diverse agrichemicals, in a single screening platform, are lacking. In this study, we examined structurally diverse agrichemicals found in the U.S. Environmental Protection Agency’s (EPA) Toxcast Phase I and II library by statically exposing early life stage zebrafish at 6 h post fertilization (hpf) until 120 hpf at concentrations ranging from 0.25 to 100 µM. Morphological outcomes were assessed at 120 hpf across 10 endpoints, including yolk sac edema, craniofacial malformations, and axis abnormalities. Chemicals that produced robust concentration-response relationships were selected for transcriptomic profiling. For transcriptomic analysis, zebrafish were statically exposed to each chemical and sampled at 48 hpf, prior to the onset of morphological effects observed at 120 hpf. Differential expression analysis identified between 0 and 4,538 differentially expressed genes (DEGs) per chemical, with no clear correlation to morphological severity. Both DEG and co-expression network analyses revealed chemical-specific expression patterns that converged on shared biological pathways, including neurodevelopment and cytoskeletal organization. Key regulatory genes such as mylpfa and krt4 were identified within co-expression modules, suggesting their potential role in conserved toxicity mechanisms. Semantic similarity analysis of enriched gene ontology (GO) terms, when compared to existing datasets, highlighted gaps in the annotation of neurodevelopmental processes, indicating that some in vivo effects may not be fully captured by current curated resources. The results provide new insights into the modes of action of diverse agrichemicals and establish a framework for understanding how agrichemical structure relates to biological function in a vertebrate model.

agrichemical↗