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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 1,459 records · Page 81
All sourcesNASA NTRSDOE OSTI
NASA NTRS1987

Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs

In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.

NASA Discipline Exobiology↗
DOE OSTI2017

Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. Furthermore, we also report non-Rif-related compounds—Nα-aroyl-N-aryl-phenylalaninamides (AAPs)—that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mechanistic modeling of in vitro transcription incorporating effects of magnesium pyrophosphate crystallization

The in vitro transcription (IVT) reaction used in the production of messenger RNA vaccines and therapies remains poorly quantitatively understood. Mechanistic modeling of IVT could inform reaction design, scale-up, and control. In this work, we develop a mechanistic model of IVT to include nucleation and growth of magnesium pyrophosphate crystals and subsequent agglomeration of crystals and DNA. To help generalize this model to different constructs, a novel quantitative description is included for the rate of transcription as a function of target sequence length, DNA concentration, and T7 RNA polymerase concentration. The model explains previously unexplained trends in IVT data and quantitatively predicts the effect of adding the pyrophosphatase enzyme to the reaction system. The model is validated on additional literature data showing an ability to predict transcription rates as a function of RNA sequence length.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Camelina circRNA landscape: Implications for gene regulation and fatty acid metabolism

Abstract Circular RNAs (circRNAs) are closed‐loop RNAs forming a covalent bond between their 3′ and 5′ ends, the back splice junction (BSJ), rendering them resistant to exonucleases and thus more stable compared to linear RNAs. Identification of circRNAs and distinction from their cognate linear RNA is only possible by sequencing the BSJ that is unique to the circRNA. CircRNAs are involved in the regulation of their cognate RNAs by increasing transcription rates, RNA stability, and alternative splicing. We have identified circRNAs from C. sativa that are associated with the regulation of germination, light response, and lipid metabolism. We sequenced light‐grown and etiolated seedlings after 5 or 7 days post‐germination and identified a total of 3447 circRNAs from 2763 genes. Most circRNAs originate from a single homeolog of the three subgenomes from allohexaploid camelina and correlate with higher ratios of alternative splicing of their cognate genes. A network analysis shows the interactions of select miRNA:circRNA:mRNAs for regulation of transcript stabilities where circRNA can act as a competing endogenous RNA. Several key lipid metabolism genes can generate circRNA, and we confirmed the presence of KASII circRNA as a true circRNA. CircRNA in camelina can be a novel target for breeding and engineering efforts.

Utley, Delecia [Department of Plant and Microbial ↗
DOE OSTI2016

A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo

hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated. To that end, we expressed an EGFP-tagged RNA binding-defective form of hnRNP K in intact Xenopus embryos, and found it was rapidly degraded in vivo. Deleting hnRNP K's nuclear localization signal (NLS), which contains two prospective ubiquitination sites, rescued the protein from degradation. These data demonstrate a novel activity for the NLS of hnRNP K in regulating the protein's stability in vivo when it is unbound to nucleic acids. - Highlights: • An RNA-binding mutant of Xenopus hnRNP K is degraded in vivo. • hnRNP K's nuclear localization signal contains two predicted ubiquitination sites. • Deletion of the nuclear localization signal rescues RNA-binding mutant degradation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. Here, a central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

High E6 Gene Expression Predicts for Distant Metastasis and Poor Survival in Patients With HPV-Positive Oropharyngeal Squamous Cell Carcinoma

Purpose: Patients with human papillomavirus (HPV)–positive oropharyngeal squamous cell carcinoma (OPSCC) have a favorable prognosis. As a result, de-escalation clinical trials are under way. However, approximately 10% of patients will experience distant recurrence even with standard-of-care treatment. Here, we sought to identify novel biomarkers to better risk-stratify HPV-positive patients with OPSCC. Methods and Materials: Gene expression profiling by RNA sequencing (RNA-seq) and quantitative polymerase chain reaction was performed on HPV-positive OPSCC primary tumor specimens from patients with and without distant metastasis (DM). Results: RNA-seq analysis of 39 HPV-positive OPSCC specimens revealed that patients with DM had 2-fold higher E6 gene expression levels than did patients without DM (P=.029). This observation was confirmed in a validation cohort comprising 93 patients with HPV-positive OPSCC. The mean normalized E6 expression level in the 17 recurring primary specimens was 13 ± 2 compared with 8 ± 1 in the remaining 76 nonrecurring primaries (P=.001). Receiver operating characteristic analysis established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). Patients from this cohort with high E6 gene expression (E6-high) (n=51, 55%) had more cancer-related deaths (23% vs 2%, P<.001) and DM (26% vs 5%, P<.001) than did patients with low E6 gene expression (E6-low) (n=42, 45%). Kaplan-Meier survival analysis revealed that E6-high had worse RFS (95% vs 69%, P=.004) and cancer-specific survival (97% vs 79%, P=.007). E6-high maintained statistical significance in multivariate regression models balancing surgery, chemotherapy, nodal stage, and smoking status. Gene set enrichment analysis demonstrated that tumors with high E6 expression were associated with P53, epidermal growth factor receptor, activating transcription factor-2, and transforming growth factor-β signaling pathways. Conclusion: High E6 gene expression level identifies HPV-positive OPSCC patients with 5-fold greater risk of distant disease recurrence and worse cancer-specific survival. Validation in a multi-institutional prospective clinical trial is required to assess the utility of E6 gene expression as a clinically useful prognostic biomarker.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2023

A landing pad system for multicopy gene integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. Furthermore, this study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Systems-Level Modeling for CRISPR-Based Metabolic Engineering

The CRISPR-Cas system has enabled the development of sophisticated, multigene metabolic engineering programs through the use of guide RNA-directed activation or repression of target genes. To optimize biosynthetic pathways in microbial systems, we need improved models to inform design and implementation of transcriptional programs. Recent progress has resulted in new modeling approaches for identifying gene targets and predicting the efficacy of guide RNA targeting. Genome-scale and flux balance models have successfully been applied to identify targets for improving biosynthetic production yields using combinatorial CRISPR-interference (CRISPRi) programs. Here, the advent of new approaches for tunable and dynamic CRISPR activation (CRISPRa) promises to further advance these engineering capabilities. Once appropriate targets are identified, guide RNA prediction models can lead to increased efficacy in gene targeting. Developing improved models and incorporating approaches from machine learning may be able to overcome current limitations and greatly expand the capabilities of CRISPR-Cas9 tools for metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Directing Nanoparticle Organization in Response to Diverse Chemical Inputs

Signaling cascades are crucial for transducing stimuli in biological systems, enabling multiple stimuli to regulate a downstream target with precisely controlled timing and amplifying signals through a series of intermediary reactions. Developing a robust signaling system with such capabilities would be pivotal for programming complex behaviors in synthetic DNA-based molecular devices. However, although “software” such as nucleic acid circuits could potentially be harnessed to relay signals to DNA-based nanostructure hardware, such explorations have been limited. Here, in this study, we develop a platform for transducing a variety of stimuli via messenger-mediated reactions to regulate the release and reloading of gold nanoparticles (AuNPs) in a 3D DNA framework. In the first step, an in vitro transcription circuit is engineered to sense and amplify chemical stimuli, including arbitrary DNA sequences and proteins, producing RNA. In the second step, the RNA releases the DNA-coated AuNPs from the DNA framework via a strand displacement reaction. AuNP reloading is controlled by a separate step driven by degradation of the RNA. Our platform holds promise for applications requiring dynamic multiagent control over DNA-based devices, offering a versatile tool for advanced molecular device engineering.

36 MATERIALS SCIENCE↗
DOE OSTI2013

Genomic properties of Marine Group A bacteria indicate a role in the marine sulfur cycle

Marine Group A (MGA) is a deeply branching and uncultivated phylum of bacteria. Although their functional roles remain elusive, MGA subgroups are particularly abundant and diverse in oxygen minimum zones and permanent or seasonally stratified anoxic basins, suggesting metabolic adaptation to oxygen-deficiency. Here, we expand a previous survey of MGA diversity in O2-deficient waters of the Northeast subarctic Pacific Ocean (NESAP) to include Saanich Inlet (SI), an anoxic fjord with seasonal O2 gradients and periodic sulfide accumulation. Phylogenetic analysis of small subunit ribosomal RNA (16S rRNA) gene clone libraries recovered five previously described MGA subgroups and defined three novel subgroups (SHBH1141, SHBH391, and SHAN400) in SI. To discern the functional properties of MGA residing along gradients of O2 in the NESAP and SI, we identified and sequenced to completion 14 fosmids harboring MGA-associated 16S RNA genes from a collection of 46 fosmid libraries sourced from NESAP and SI waters. Comparative analysis of these fosmids, in addition to four publicly available MGA-associated large-insert DNA fragments from Hawaii Ocean Time-series and Monterey Bay, revealed widespread genomic differentiation proximal to the ribosomal RNA operon that did not consistently reflect subgroup partitioning patterns observed in 16S rRNA gene clone libraries. Predicted protein-coding genes associated with adaptation to O2-deficiency and sulfur-based energy metabolism were detected on multiple fosmids, including polysulfide reductase (psrABC), implicated in dissimilatory polysulfide reduction to hydrogen sulfide and dissimilatory sulfur oxidation. These results posit a potential role for specific MGA subgroups in the marine sulfur cycle.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

The structure of Zika virus NS5 reveals a conserved domain conformation

The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. In conclusion, the activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antivirals for ZIKV.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Impact of wet-dry cycling on the phase behavior and compartmentalization properties of complex coacervates

Abstract Wet-dry cycling on the early Earth is thought to have facilitated production of molecular building blocks of life, but its impact on self-assembly and compartmentalization remains largely unexplored. Here, we investigate dehydration/rehydration of complex coacervates, which are membraneless compartments formed by phase separation of polyelectrolyte solutions. Solution compositions are identified for which tenfold water loss results in maintenance, disappearance, or appearance of coacervate droplets. Systems maintaining coacervates throughout the dehydration process are further evaluated to understand how their compartmentalization properties change with drying. Although added total RNA concentrations increase tenfold, RNA concentration within coacervates remains steady. Exterior RNA concentrations rise, and exchange rates for encapsulated versus free RNAs increase with dehydration. We explain these results in light of the phase diagram, with dehydration-driven ionic strength increase being particularly important in determining coacervate properties. This work shows that wet-dry cycling can alter the phase behavior and protocell-relevant functions of complex coacervates.

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

Molecular mechanisms underlying the extreme mechanical anisotropy of the flaviviral exoribonuclease-resistant RNAs (xrRNAs)

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using a single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg 2+ and the key tertiary interactions. The absence of Mg 2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structures in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations in combination with force distribution analysis. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid–specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2'-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Developmental assembly of multi-component polymer systems through interconnected synthetic gene networks in vitro

Abstract Living cells regulate the dynamics of developmental events through interconnected signaling systems that activate and deactivate inert precursors. This suggests that similarly, synthetic biomaterials could be designed to develop over time by using chemical reaction networks to regulate the availability of assembling components. Here we demonstrate how the sequential activation or deactivation of distinct DNA building blocks can be modularly coordinated to form distinct populations of self-assembling polymers using a transcriptional signaling cascade of synthetic genes. Our building blocks are DNA tiles that polymerize into nanotubes, and whose assembly can be controlled by RNA molecules produced by synthetic genes that target the tile interaction domains. To achieve different RNA production rates, we use a strategy based on promoter “nicking” and strand displacement. By changing the way the genes are cascaded and the RNA levels, we demonstrate that we can obtain spatially and temporally different outcomes in nanotube assembly, including random DNA polymers, block polymers, and as well as distinct autonomous formation and dissolution of distinct polymer populations. Our work demonstrates a way to construct autonomous supramolecular materials whose properties depend on the timing of molecular instructions for self-assembly, and can be immediately extended to a variety of other nucleic acid circuits and assemblies.

Science & Technology - Other Topics↗
DOE OSTI2025

Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates

The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.

59 BASIC BIOLOGICAL SCIENCES↗