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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 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Quantum Light Emission from Coupled Defect States in DNA-Functionalized Carbon Nanotubes

Solid-state single-photon sources are essential building blocks for quantum photonics and quantum information technologies. This study demonstrates promising single-photon emission from quantum defects generated in single-wall carbon nanotubes (SWCNTs) by covalent reaction with guanine nucleotides in their single-stranded DNA coatings. Low-temperature photoluminescence spectroscopy and photon-correlation measurements on individual guanine-functionalized SWCNTs (GF-SWCNTs) indicate that multiple, closely spaced guanine defect sites within a single ssDNA strand collectively form an exciton trapping potential that supports a localized quantum state capable of room-temperature single-photon emission. In addition, exciton traps from adjacent ssDNA strands are weakly coupled to give cross-correlations between their separate photon emissions. Theoretical modeling identifies coupling mechanism as a capture of band-edge excitons. Finally, because the spatial pattern of nanotube functionalization sites can be readily controlled by selecting ssDNA base sequences, GF-SWCNTs should become a versatile family of quantum light emitters with engineered properties.

36 MATERIALS SCIENCE↗
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

SPLIT: Stable Protein Coacervation Using a Light Induced Transition

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellularsized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. Finally, the methods described here provide novel strategies for inducing protein phase separation using light.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2021

Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell genomic resolution

With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek's Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors

Abstract Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum , ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural basis of RNA cap modification by SARS-CoV-2

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A deep learning approach to programmable RNA switches

Engineered RNA elements are programmable tools capable of detecting small molecules, proteins, and nucleic acids. Predicting the behavior of these synthetic biology components remains a challenge, a situation that could be addressed through enhanced pattern recognition from deep learning. Here, we investigate Deep Neural Networks (DNN) to predict toehold switch function as a canonical riboswitch model in synthetic biology. To facilitate DNN training, we synthesize and characterize in vivo a dataset of 91,534 toehold switches spanning 23 viral genomes and 906 human transcription factors. DNNs trained on nucleotide sequences outperform (R 2 = 0.43–0.70) previous state-of-the-art thermodynamic and kinetic models (R 2 = 0.04–0.15) and allow for human-understandable attention-visualizations (VIS4Map) to identify success and failure modes. This work shows that deep learning approaches can be used for functionality predictions and insight generation in RNA synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Selective inhibition of human translation termination by a drug-like compound

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein—dephosphorylation of deoxynucleotide triphosphates (dNTPs)—implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3’ to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase

Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5' end of the RNA genome as a promoter for RNA synthesis. Flaviviral polymerase NS5 specifically recognizes SLA to initiate RNA synthesis and methylate the 5' guanosine cap. We report the crystal structures of dengue (DENV) and Zika virus (ZIKV) SLAs. DENV and ZIKV SLAs differ in the relative orientations of their top stem-loop helices to bottom stems, but both form an intermolecular three-way junction with a neighboring SLA molecule. To understand how NS5 engages SLA, we determined the SLA-binding site on NS5 and modeled the NS5-SLA complex of DENV and ZIKV. Our results show that the gross conformational differences seen in DENV and ZIKV SLAs can be compensated by the differences in the domain arrangements in DENV and ZIKV NS5s. We describe two binding modes of SLA and NS5 and propose an SLA-mediated RNA synthesis mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease

ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

In crystallo observation of three metal ion promoted DNA polymerase misincorporation

Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds–Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly

Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain ( spb 1 D52A ) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF 4 - transition state structure that implicates the direct involvement of unmodified G2922 in Nog2 GTPase activation. Genetic suppressors and in vivo imaging indicate that premature GTP hydrolysis prevents the efficient binding of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 recruitment to the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Our approach and findings provide a template to study the GTPase cycles and regulatory factor interactions of the other K-loop GTPases involved in ribosome assembly.

59 BASIC BIOLOGICAL SCIENCES↗