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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 OSTI2024

A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain

Abstract SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5′-TCGGAT-3′) of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases

The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. Here, to address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an) and further trace Cas13 origins to defense-associated ribonucleases. Despite being one-third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing that the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A conserved viral RNA fold enables nuclease resistance across kingdoms of life

Abstract Viral exoribonuclease-resistant RNA (xrRNA) structures block cellular nucleases to produce subgenomic viral RNAs during infection. High sequence variability among xrRNAs from distantly related viruses raises questions about the shared molecular features that enable these RNAs to withstand the strong unwinding forces of exoribonucleases. Here, we present the first structure of a plant-virus xrRNA in its active conformation and uncover universal principles of xrRNA folding. Comparison with the structure of a human-pathogenic flavivirus xrRNA reveals that both share a core structural motif—a protective ring encircling the RNA’s 5′ end—despite lacking sequence similarity. Disrupting this core motif through targeted mutagenesis eliminates exoribonuclease-resistance and attenuates viral infection. We identify hundreds of related structures across multiple virus families, supporting the conservation of this mechanism. Our study demonstrates how distantly related RNA viruses have converged on a common structural strategy to inhibit cellular nucleases, with a universal ring topology as the defining feature of viral xrRNAs.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Expansion of the tmRNA sequence database and new tools for search and visualization

Abstract Transfer–messenger RNA (tmRNA) contributes essential tRNA-like and mRNA-like functions during the process of trans-translation, a mechanism of quality control for the translating bacterial ribosome. Proper tmRNA identification benefits the study of trans-translation and also the study of genomic islands, which frequently use the tmRNA gene as an integration site. Automated tmRNA gene identification tools are available, but manual inspection is still important for eliminating false positives. We have increased our database of precisely mapped tmRNA sequences over 50-fold to 97 179 unique sequences. Group I introns had previously been found integrated within a single subsite within the TψC-loop; they have now been identified at four distinct subsites, suggesting multiple founding events of invasion of tmRNA genes by group I introns, all in the same vicinity. tmRNA genes were found in metagenomic archaeal genomes, perhaps a result of misbinning of bacterial sequences during genome assembly. With the expanded database, we have produced new covariance models for improved tmRNA sequence search and new secondary structure visualization tools.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Diaminopurine in Nonenzymatic RNA Template Copying

In the RNA World before the emergence of an RNA polymerase, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides (A, U, C, and G) are heavily biased toward the incorporation of G and C, which form a more stable base pair than A and U. We therefore asked whether replacing adenine (A) with diaminopurine (D) might lead to more efficient and less biased nonenzymatic template copying by making a stronger version of the A:U pair. As expected, primer extension substrates containing D bound to U in the template more tightly than substrates containing A. However, primer extension with D exhibited elevated reaction rates on a C template, leading to concerns about fidelity. Our crystallographic studies revealed the nature of the D:C mismatch by showing that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease the mismatched primer extension. We performed nonenzymatic primer extension with all four activated nucleotides on randomized RNA templates containing all four letters and used deep sequencing to analyze the products. We found that the DUCG genetic system exhibited a more even product distribution and a lower mismatch frequency than the canonical AUCG system. Furthermore, primer extension is greatly reduced following all mismatches, including the D:C mismatch. Our study suggests that D deserves further attention for its possible role in the RNA World and as a potentially useful component of artificial nonenzymatic RNA replication systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Multiplexed CRISPR-Cas9 mutagenesis of rice PSBS1 noncoding sequences for transgene-free overexpression

Understanding CRISPR-Cas9’s capacity to produce native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR-Cas9 mutagenesis of noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo—from knockout to overexpression—using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5'UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, evidence the potential of CRISPR-Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Viroid-like “obelisk” agents are widespread in the ocean and exceed the abundance of RNA viruses in the prokaryotic fraction

Abstract “Obelisks” are recently discovered ribonucleic acid (RNA) viroid-like elements present in diverse environments with no phylogenetic similarity to any known biological agent. obelisks were first identified in the human gut and in a commensal bacterium acting as a replicative host. They have a circular ∼1 kb RNA genome, rod-like secondary structures, and the encoding of a protein superfamily called “Oblins”. We performed a large-scale search of obelisks in the ocean using the Pebblescout program and the transcriptomic Sequence Archive Read databases, revealing the biogeography and abundance of these viroid-like RNA elements. We detected 55 obelisk genomes resulting in 35 marine clusters at the species level. These obelisks were detected in the prokaryotic fraction and to a lesser extent in the eukaryotic fraction, and distributed across all the oceans from surface to mesopelagic including the Arctic, and even in the coldest seawater of Earth beneath the Antarctic Ross Ice Shelf. The obelisk hallmark protein Oblin-1 confirmed by 3D models was found in various marine samples. Some of the detected marine obelisks harbor hammerhead self-cleaving ribozymes in both polarities. In the prokaryotic, but not the eukaryotic, fraction of the Tara Ocean dataset, relative abundance of obelisks calculated by transcriptomic fragment recruitment indicated that they are abundant in marine samples, reaching or even exceeding the relative abundance of the previously discovered uncultured RNA viruses. In conclusion, obelisks are abundant and widespread viroid-like elements that should be included in ocean biogeochemical models.

Environmental Sciences & Ecology↗
DOE OSTI2026

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗
DOE OSTI2025

Taxogenomic analysis of Pichia senei sp. nov. and new insights into hybridization events in the Pichia cactophila species complex

Three strains of a novel yeast species were isolated from necrotic cactus tissues of Cereus saddianus and Micranthocereus dolichospermaticus and from phytotelmata of Bromelia karatas. DNA sequence analysis of the Internal Transcribed Spacer (ITS) region and D1/D2 domains of the large subunit ribosomal RNA, along with whole genome phylogenomic analysis, showed that this yeast is most closely related to Pichia insulana, Pichia cactophila, and Pichia inconspicua. The new species differs by 10–13 nucleotide substitutions from these species in D1/D2 sequences and exhibits <90% genome-wide average nucleotide identity to them. The name Pichia senei sp. nov. is proposed for the novel species, which is homothallic and produces asci with one to four hat-shaped ascospores. The holotype is CBS 16311 (MycoBank MB 858723). Taxogenomic analyses of the P. cactophila species complex, including P. senei, provide new insights about the hybridizations events that shaped this group. Pichia insulana and P. inconspicua are identified as the parental lineages that originated P. cactophila, and P. senei also appears closely related to one of the progenitors of P. inconspicua. We assess phylogeny, heterozygosity, and ploidy to explore the processes shaping diversity, showing how genomic data support yeast species delimitation and reveal complex hybridization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Unraveling plant–microbe symbioses using single-cell and spatial transcriptomics

Plant-microbe symbioses require intense interaction and genetic coordination to successfully establish in specific cell types of the host and symbiont. Traditional RNA-seq methodologies lack the cellular resolution to fully capture these complexities, but single-cell and spatial transcriptomics (ST) are now allowing scientists to probe symbiotic interactions at an unprecedented level of detail. Here, we discuss the advantages that novel spatial and single-cell transcriptomic technologies provide in studying plant-microbe endosymbioses and highlight key recent studies. Finally, we consider the remaining limitations of applying these approaches to symbiosis research, which are mainly related to the simultaneous capture of both plant and microbial transcripts within the same cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Naturally ornate RNA-only complexes revealed by cryo-EM

The structures of natural RNAs remain poorly characterized and may hold numerous surprises. Here we report three-dimensional structures of three large ornate bacterial RNAs using cryo-electron microscopy (cryo-EM). GOLLD (Giant, Ornate, Lake- and Lactobacillales-Derived), ROOL (Rumen-Originating, Ornate, Large) and OLE (Ornate Large Extremophilic) RNAs form homo-oligomeric complexes whose stoichiometries are retained at lower concentrations than measured in cells. OLE RNA forms a dimeric complex with long co-axial pipes spanning two monomers. Both GOLLD and ROOL form distinct RNA-only multimeric nanocages with diameters larger than the ribosome, each empty except for a disordered loop. Extensive intramolecular and intermolecular A-minor interactions, kissing loops, an unusual A–A helix and other interactions stabilize the three complexes. Sequence covariation analysis of these large RNAs reveals evolutionary conservation of intermolecular interactions, supporting the biological importance of large, ornate RNA quaternary structures that can assemble without any involvement of proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Sequence-specific dynamic DNA bending explains mitochondrial TFAM’s dual role in DNA packaging and transcription initiation

Abstract Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent—LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Development of high throughput and in vitro assays for analyzing RNA modifications

Modifications on RNAs play major roles in their stability, translation, and enzymatic activity. Despite its importance, the current techniques are insufficient to study the structure and function of RNA modifications. Indeed, the National Academies of Science, Engineering and Medicine indicate that developing new tools and further study the function of RNA modifications is strategically a high priority for advancing science in the coming years (https://www.nationalacademies.org/our-work/toward-sequencing-and-mapping-of-rna-modifications). RNA modifications occur in all domains of life controlling processes such as RNA turnover, translation regulation, cellular defenses and bioproduction. Our preliminary data indicated that the insulin mRNA might get ADP-ribosylated by the ADP-ribosyltransferase PARP12. RNA ADP-ribosylation has been described in Escherichia coli. Combined to the fact that ADP-ribosyltransferase (PARP) genes are conserved throughout evolution we hypothesize that this modification might play essential roles in cells. Therefore, we proposed to develop sequencing techniques and in vitro enzymatic assays to identify and validate ADP-ribosylation motifs and sites. Here we report the development of RNA-seq and qPCR assays to identify ADP-ribosylated RNAs, in addition to a nicotinamide adenosine dinucleotide (NAD – ADP-ribosylation donor) consumption assay and an enzyme-linked immunosorbent assay (ELISA) to measure ADP-ribosyltransferase activity. Testing these assays with the insulin mRNA confirmed that this transcript is ADP-ribosylated. These assays will not only enable studying the function of ADP-ribosylation but can be easily adapted for studying other RNA modifications. This will open opportunities to study RNA modifications in different model systems from bacteria to viruses to plants, bringing insights into their cellular functions and the possibility of targeting them for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
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↗
DOE OSTI2026

A tunable autonomous RNA-fueled micro-engine

Autonomous molecular machines capable of converting chemical energy into mechanical motion are foundational components for synthetic nanoscale systems. Inspired by biological motors, we report the construction of a tunable, RNA-fueled DNA origami engine that drives the cyclic movement of a 500 nm-diameter particle at the microscale. The engine operates via sequential RNA–DNA hybridization and enzymatic cleavage by RNase H, enabling reversible switching between folded and unfolded conformations without external intervention. By modulating RNA and enzyme concentrations and controlling temperature, we achieve tunable switching kinetics, with transition periods as short as ~10 s. Kinetic modeling reveals that the folding pathway is governed by both productive RNA binding and the enzymatic clearance of misfolded intermediates, while unfolding is primarily controlled by RNase H activity. Since the RNA fuel binds specifically to the DNA strands, each engine is addressable simply by changing the sequences. This work demonstrates a programmable, self-resetting molecular actuator and offers a blueprint for building more complex nanomechanical systems with forces and energies comparable to molecular motors.

DNA nanomachines↗
DOE OSTI2024

Binding profiles for 961 Drosophila and C. elegans transcription factors reveal tissue-specific regulatory relationships

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here, we present the culmination of the efforts of the modENCODE (model organism Encyclopedia of DNA Elements) and modERN (model organism Encyclopedia of Regulatory Networks) consortia to systematically assay TF binding events in vivo in two major model organisms,Drosophila melanogaster(fly) andCaenorhabditis elegans(worm). These data sets comprise 605 TFs identifying 3.6 M sites in the fly and 356 TFs identifying 0.9 M sites in the worm, and represent the majority of the regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters termed “metapeaks,” that larger metapeaks have characteristics of high-occupancy target (HOT) regions, and that the importance of consensus sequence motifs bound by TFs depends on metapeak size and complexity. Combining ChIP-seq data with single-cell RNA-seq data in a machine-learning model identifies TFs with a prominent role in promoting target gene expression in specific cell types, even differentiating between parent–daughter cells during embryogenesis. These data are a rich resource for the community that should fuel and guide future investigations into TF function. To facilitate data accessibility and utility, all strains expressing green fluorescent protein (GFP)-tagged TFs are available at the stock centers for each organism. The chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center, GEO, and through a direct interface that provides rapid access to processed data sets and summary analyses, as well as widgets to probe the cell-type-specific TF–target relationships.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Transcriptomics outputs and phylogenetic trees used for pathway discovery of diterpenoid alkaloids in Delphinium and Aconitum

Transcriptome assemblies, open reading frames in nucleotide and peptide sequences, clustered transcriptomes and corresponding amino acid files, and expression matrices in TPM and raw counts for RNA-seq datasets from Delphinium grandiflorum, Aconitum plicatum, Aconitum lycoctonum, Aconitum carmichaelii, Aconitum japonicum, Aconitum kusnezoffii, and Aconitum vilmorinianum. Also included are phylogenetic trees for terpene synthases and cytochromes P450 mined from these assemblies.

biosynthesis↗
DOE OSTI2026

Impact of Selenium and Vitamin E Deficiency on Zika Virus Pathogenesis and Immune Response in Mice

Micronutrient status is recognized to influence host susceptibility to viral infections, yet its impact on Zika virus (ZIKV) pathogenesis remains incompletely understood. We investigated the effects of dietary selenium and combined selenium plus vitamin E deficiency on ZIKV infection outcomes in a type I interferon α/β receptor knockout (Ifnar1 −/− ) murine model. Mice maintained on deficient diets exhibited significantly lower neutralizing antibody titers and reduced levels of key antiviral cytokines (IFN-γ, TNF-α, IFN-α, IFN-β, IL-12p70, CCL5) compared to controls. Correspondingly, higher viral RNA loads were detected in the brains of double-deficient mice, which also experienced greater weight loss and increased mortality. Deep sequencing revealed no major differences in overall viral genome diversity across diet groups; however, specific mutations, including V330L and D67E in the E gene, and V360I in the NS3 gene, were enriched or detected in nutritionally deficient animals. These findings suggest that antioxidant micronutrient deficiency impairs both humoral and cellular immune responses to ZIKV, potentially facilitating enhanced neuroinvasion. While the functional consequences of the identified mutations warrant further investigation, our results underscore the importance of adequate micronutrient intake for optimal antiviral defense. Further studies are needed to clarify the epidemiological significance of these observations.

Biological and medical sciences↗