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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 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp 215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Science & Technology - Other Topics↗
DOE OSTI2024

Structural basis for a highly conserved RNA-mediated enteroviral genome replication

Abstract Enteroviruses contain conserved RNA structures at the extreme 5′ end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3′ spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3′ spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Self-assembly and condensation of intermolecular poly(UG) RNA quadruplexes

Abstract Poly(UG) or ‘pUG’ dinucleotide repeats are highly abundant sequences in eukaryotic RNAs. In Caenorhabditis elegans, pUGs are added to RNA 3′ ends to direct gene silencing within Mutator foci, a germ granule condensate. Here, we show that pUG RNAs efficiently self-assemble into gel condensates through quadruplex (G4) interactions. Short pUG sequences form right-handed intermolecular G4s (pUG G4s), while longer pUGs form left-handed intramolecular G4s (pUG folds). We determined a 1.05 Å crystal structure of an intermolecular pUG G4, which reveals an eight stranded G4 dimer involving 48 nucleotides, 7 different G and U quartet conformations, 7 coordinated potassium ions, 8 sodium ions and a buried water molecule. A comparison of the intermolecular pUG G4 and intramolecular pUG fold structures provides insights into the molecular basis for G4 handedness and illustrates how a simple dinucleotide repeat sequence can form complex structures with diverse topologies.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer

Abstract Tagging RNAs with fluorogenic aptamers has enabled imaging of transcripts in living cells, revealing novel aspects of RNA metabolism and dynamics. While a diverse set of fluorogenic aptamers has been developed, a new generation of aptamers uses the ring-opening of spirocyclic rhodamine dyes to achieve robust performance in live mammalian cells. These fluorophores have two chemical states: a cell-permeable spirocyclic state and a fluorescent zwitterionic state. The SpyRho555 dye is mostly non-fluorescent in solution and becomes fluorescent in complex with the RhoBAST aptamer. To understand the basis for RhoBAST-SpyRho555 fluorogenicity, we have determined crystal structures of RhoBAST in complex with 5-carboxytetramethylrhodamine and a SpyRho555 analogue, MaP555. RhoBAST is organized by a perfect four-way junction that positions two loops to form the dye-binding pocket. The core of the ligand resides between a tri-adenine floor and a single guanine base, largely driven by π-stacking interactions. Importantly, the unpaired guanine interacts with the 3-position group of MaP555 to stabilize the open conformation, supported by mutagenesis data, and may play an active role in promoting the open conformation of the dye. This work has implications for the development of new fluorogenic aptamers with improved properties using structure-guided design approaches.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Dual-RNA-sequencing to elucidate the interactions between sorghum and Colletotrichum sublineola

In warm and humid regions, the productivity of sorghum is significantly limited by the fungal hemibiotrophic pathogen Colletotrichum sublineola , the causal agent of anthracnose, a problematic disease of sorghum ( Sorghum bicolor (L.) Moench) that can result in grain and biomass yield losses of up to 50%. Despite available genomic resources of both the host and fungal pathogen, the molecular basis of sorghum− C. sublineola interactions are poorly understood. By employing a dual-RNA sequencing approach, the molecular crosstalk between sorghum and C. sublineola can be elucidated. In this study, we examined the transcriptomes of four resistant sorghum accessions from the sorghum association panel (SAP) at varying time points post-infection with C. sublineola . Approximately 0.3% and 93% of the reads mapped to the genomes of C. sublineola and Sorghum bicolor , respectively. Expression profiling of in vitro versus in planta C. sublineola at 1-, 3-, and 5-days post-infection (dpi) indicated that genes encoding secreted candidate effectors, carbohydrate-active enzymes (CAZymes), and membrane transporters increased in expression during the transition from the biotrophic to the necrotrophic phase (3 dpi). The hallmark of the pathogen-associated molecular pattern (PAMP)-triggered immunity in sorghum includes the production of reactive oxygen species (ROS) and phytoalexins. The majority of effector candidates secreted by C. sublineola were predicted to be localized in the host apoplast, where they could interfere with the PAMP-triggered immunity response, specifically in the host ROS signaling pathway. The genes encoding critical molecular factors influencing pathogenicity identified in this study are a useful resource for subsequent genetic experiments aimed at validating their contributions to pathogen virulence. This comprehensive study not only provides a better understanding of the biology of C. sublineola but also supports the long-term goal of developing resistant sorghum cultivars.

Vela, Saddie↗
DOE OSTI2024

CrossMP: Enabling Cross-Modality Translation between Single-Cell RNA-Seq and Single-Cell ATAC-Seq through Web-Based Portal

In recent years, there has been a growing interest in profiling multiomic modalities within individual cells simultaneously. One such example is integrating combined single-cell RNA sequencing (scRNA-seq) data and single-cell transposase-accessible chromatin sequencing (scATAC-seq) data. Integrated analysis of diverse modalities has helped researchers make more accurate predictions and gain a more comprehensive understanding than with single-modality analysis. However, generating such multimodal data is technically challenging and expensive, leading to limited availability of single-cell co-assay data. Here, we propose a model for cross-modal prediction between the transcriptome and chromatin profiles in single cells. Our model is based on a deep neural network architecture that learns the latent representations from the source modality and then predicts the target modality. It demonstrates reliable performance in accurately translating between these modalities across multiple paired human scATAC-seq and scRNA-seq datasets. Additionally, we developed CrossMP, a web-based portal allowing researchers to upload their single-cell modality data through an interactive web interface and predict the other type of modality data, using high-performance computing resources plugged at the backend.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Conditional guide RNA deactivation by mRNA and small molecule triggers in Saccharomyces cerevisiae

CRISPR interference (CRISPRi) technologies have revolutionized bioengineering by providing precise tools for gene expression modulation, enabling targeted gene perturbation and metabolic pathway optimization. Despite these advances, achieving dynamic control over gene expression by CRISPR-based regulation remains a challenge due to its inherently static nature. Utilizing toehold-mediated strand displacement and ligand-responsive ribozymes (aptazymes), this study introduces switchable guide RNAs (gRNAs) that facilitate tunable gene expression mediated by mRNA or small molecule signals. We demonstrate complete silencing of gRNA via strategically designed 5’ or 3’ extensions that impede the gRNA spacer or the dCas9 handle, with subsequent restoration of function through sequestration or cleavage of the obstructive sequence. The resulting toehold-embedded or aptazyme-embedded gRNAs can be deactivated by specific signals, including two full-length translatable mRNAs and two small molecule triggers, thereby lifting CRISPRi repression on targeted genes. This modular approach allows for gRNA-based biocomputing through multi-layer or multi-input genetic logic gates in Saccharomyces cerevisiae . Offering a versatile strategy for post-CRISPR regulation in response to environmental signals or cellular states, this methodology expands the toolkit in eukaryotic systems for reversible control of gene expression.

Aptazyme↗
DOE OSTI2024

RNA-seq and metabolomic analyses of beneficial plant phenol biochemical pathways in red alder

Red alder ( Alnus rubra ) has highly desirable wood, dye pigment, and (traditional) medicinal properties which have been capitalized on for thousands of years, including by Pacific West Coast Native Americans. A rapidly growing tree species native to North American western coastal and riparian regions, it undergoes symbiosis with actinobacterium Frankia via their nitrogen-fixing root nodules. Red alder’s desirable properties are, however, largely attributed to its bioactive plant phenol metabolites, including for plant defense, for its attractive wood and bark coloration, and various beneficial medicinal properties. Integrated transcriptome and metabolome data analyses were carried out using buds, leaves, stems, roots, and root nodules from greenhouse grown red alder saplings with samples collected during different time-points (Spring, Summer, and Fall) of the growing season. Pollen and catkins were collected from field grown mature trees. Overall plant phenol biochemical pathways operative in red alder were determined, with a particular emphasis on potentially identifying candidates for the long unknown gateway entry points to the proanthocyanidin (PA) and ellagitannin metabolic classes, as well as in gaining better understanding of the biochemical basis of diarylheptanoid formation, i.e. that help define red alder’s varied medicinal uses, and its extensive wood and dye usage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Integrated multi-omic characterizations of the synapse reveal RNA processing factors and ubiquitin ligases associated with neurodevelopmental disorders

The molecular composition of the excitatory synapse is incompletely defined due to its dynamic nature across developmental stages and neuronal populations. To address this gap, we apply proteomic mass spectrometry to characterize the synapse in multiple biological models including the fetal human brain and hiPSC-derived neurons. To prioritize the identified proteins, we develop an orthogonal multi-omic screen of genomic, transcriptomic, interactomic, and structural data. This data-driven framework identifies proteins with key molecular features intrinsic to the synapse, including characteristic patterns of biophysical interactions and cross-tissue expression. The multi-omic analysis captures synaptic proteins across developmental stages and experimental systems, including 493 synaptic candidates supported by proteomics. We further investigate three such proteins that are associated with neurodevelopmental disorders – the CUL3 E3 ubiquitin ligase, the DDX3X and YBX1 nucleic-acid binding proteins – by mapping their networks of physically interacting synapse proteins or transcripts. Our study demonstrates the potential of an integrated multi-omic approach to systematically and more comprehensively resolve the synaptic architecture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Structural Analysis of 23S rRNA Methylating Enzyme Cfr Reveals RNA-Binding Determinants for Methylation Regiospecificity and Antibiotic Resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli, and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPS A phenotype). Cfr catalyzes methylation of the C8 position of the A2503 base in 23S rRNA, the recognition site of the above antibiotic classes. Along with the RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfr’s dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb PhLOPS A resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in cellulo 23S rRNA methylated species, and in cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multidrug resistance activity. In particular, we found that Cfr’s Q329 residue is important for C8- specific methylation. These data provide a framework for further studies of the biochemistry, structure, and inhibition of this important resistance determinant.

AlphaFold↗