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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 613 records · Page 34
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Parallel Guanine Duplex and Cytosine Duplex DNA with Uninterrupted Spines of Ag I -Mediated Base Pairs

Here, hydrogen bonding between nucleobases produces diverse DNA structural motifs, including canonical duplexes, guanine (G) quadruplexes, and cytosine (C) i-motifs. Incorporating metal-mediated base pairs into nucleic acid structures can introduce new functionalities and enhanced stabilities. Here we demonstrate, using mass spectrometry (MS), ion mobility spectrometry (IMS), and fluorescence resonance energy transfer (FRET), that parallel-stranded structures consisting of up to 20 G–Ag I –G contiguous base pairs are formed when natural DNA sequences are mixed with silver cations in aqueous solution. FRET indicates that duplexes formed by poly(cytosine) strands with 20 contiguous C–Ag I –C base pairs are also parallel. Silver-mediated G-duplexes form preferentially over G-quadruplexes, and the ability of Ag + to convert G-quadruplexes into silver-paired duplexes may provide a new route to manipulating these biologically relevant structures. IMS indicates that G-duplexes are linear and more rigid than B-DNA. DFT calculations were used to propose structures compatible with the IMS experiments. Such inexpensive, defect-free, and soluble DNA-based nanowires open new directions in the design of novel metal-mediated DNA nanotechnology.

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

Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi -Containing Consortium SDC-9

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). In this work, metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13 C-/ 15 N-labeled peptides determined 1.8 × 10 3 TceA and 1.2 × 10 2 VcrA RDase molecules per Dhc cell. Ultimately, the MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Quantitative Proteomics Analysis of Camelina sativa Seeds Overexpressing the AGG3 Gene to Identify the Proteomic Basis of Increased Yield and Stress Tolerance

Camelina sativa , a close relative of Arabidopsis , is an oilseed plant that is emerging as an important biofuel resource. The genome and transcriptome maps of Camelina have become available recently, but its proteome composition remained unexplored. In this work, a labeling LC-based quantitative proteomics approach was applied to decipher the Camelina seed proteome, which led to the identification of 1532 proteins. In addition, the effect of overexpression of the Arabidopsis G-protein γ subunit 3 ( AGG3 ) on the Camelina seed proteome was elucidated to identify the proteomic basis of its increased seed size and improved stress tolerance. The comparative analysis showed a significantly higher expression of proteins involved in primary and secondary metabolism, nucleic acid and protein metabolism, and abscisic acid related responses, corroborating the physiological effects of AGG3 overexpression. More importantly, the proteomic data suggested involvement of the AGG3 protein in the regulation of oxidative stress and heavy metal stress tolerance. These observations were confirmed by the physiological and biochemical characterization of AGG3 -overexpressing seeds, which exhibit a higher tolerance to exogenous cadmium in a glutathione-dependent manner. The activity of multiple redox-regulating enzymes is higher in seeds expressing enhanced levels of AGG3 . Overall, these data provide critical evidence for the role of redox regulation by the AGG3 protein in mediating important seed-related traits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

3D Columnar Phase of Stacked Short DNA Organized by Coherent Membrane Undulations

We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (d sDNA ) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing d sDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA–DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent “crystal-like” undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large d sDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (∝ κh 2 /d sDNA 4 , h 2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. Here, the findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bottom-Up Simulation, Reconstruction, and Quantification of Macromolecule Sequences from Experimental Polymerizations

Motivated by the canonical sequence–structure–function paradigm, tools to characterize chemical patterning in natural biomacromolecules, from proteins to nucleic acids, have grown exponentially in recent years. However, analogous strategies for synthetic macromolecules remain in nascent stages, complicated by sequence polydispersity and analytical limitations. To address this, we have developed a comprehensive and open-source Python package, PRISM (polymer rate insights and sequence modeling), an end-to-end workflow that provides a path from experimental kinetics measurements to quantitative and qualitative metrics for describing chemical patterning in stochastic polymers. First, a numerical integration strategy was constructed to simulate and fit experimental data from reversible addition–fragmentation chain transfer (RAFT) polymerization kinetics, enabling the facile estimation of relevant reactivity ratios. These ratios were then used in a mechanism-specific stochastic kinetic simulation strategy to simulate sequence ensembles corresponding to model systems spanning experimental copolymers, classes of statistical polymers (e.g., alternating, block, and gradient), and multiblock copolymers. Lastly, inspired by sequence homology metrics from bioinformatics, we introduce visualization strategies and quantitative metrics to facilitate comparisons of different sequence ensembles. As the sequence–structure–function paradigm becomes increasingly central in de novo design of synthetic macromolecules, this toolkit provides a first step toward accurate and representative sequence description and featurization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Molecular Basis for Autocatalytic Backbone N -Methylation in RiPP Natural Product Biosynthesis

N-methylation of nucleic acids, proteins, and peptides is a chemical modification with significant impact on biological regulation. Despite the simplicity of the structural change, N-methylation can influence diverse functions including epigenetics, protein complex formation, and microtubule stability. While there are limited examples of N-methylation of the α-amino group of bacterial and eukaryotic proteins, there are no examples of catalysts that carry out post-translation methylation of backbone amides in proteins or peptides. Recent studies have identified enzymes that catalyze backbone N-methylation on a peptide substrate, a reaction with little biochemical precedent, in a family of ribosomally synthesized natural products produced in basidiomycetes. Here, we describe the crystal structures of Dendrothele bispora dbOphMA, a methyltransferase that catalyzes multiple N-methylations on the peptide backbone. We further carry out biochemical studies of this catalyst to determine the molecular details that promote this unusual chemical transformation. As a result, the structural and biochemical framework described here could facilitate biotechnological applications of catalysts for the rapid production of backbone N-methylated peptides.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Tre-DST: A Drug Susceptibility Test for Mycobacterium tuberculosis Using Solvatochromic Trehalose Probes

In 2024, an estimated 10 million people developed Tuberculosis (TB), nearly half a million of whom were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance enables rapid engagement in effective care. Bacterial culture and nucleic acid testing remain the primary diagnostic methods, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance, such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel metabolically incorporated trehalose probes, which specifically detect live mycobacteria. We used the nonpathogenic Mycobacterium smegmatis and the virulence-attenuated Mycobacterium tuberculosis (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting that the plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a 1-week incubation step allowed Mtb samples originally seeded at 10 4 CFU/mL to become detectable, over 2 weeks earlier than colony-forming unit analysis. We found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs as well as the newer drug bedaquiline. Tre-DST distinguished RIF- and INH-resistant auxotrophs from susceptible controls and accurately reported the resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended and future DR-TB drugs as a diagnostic in reference laboratories.

diagnostics↗
DOE OSTI2025

Nanotubes Growth by Self-Assembly of DNA Strands at Room Temperature

Artificial biomolecular nanotubes are a promising approach to building materials mimicking the capacity of the cellular cytoskeleton to grow and self-organize dynamically. Nucleic acid nanotechnology has demonstrated a variety of self-assembling nanotubes with programmable, robust features and morphological similarities to actual cytoskeleton components. However, their production typically requires thermal annealing, which not only poses a general constraint on their potential applications but is also incompatible with physiological conditions. Here, we demonstrate that DNA nanotubes can self-assemble from a simple mixture of five short DNA strands at constant room temperature, growing for extended periods of time in bulk conditions as well as under confinement. Assembly is achieved using a monovalent salt buffer, which ensures a faithful nanoscale arrangement and avoids nanotube aggregation. We observe the formation of individual nanotubes up to 20 days with a diameter of 22 ± 4 nm and length of several tens of micrometers. We finally encapsulate the strands in microsized compartments, such as water-in-oil microdroplets and giant unilamellar vesicles serving as simple cell models. Notably, nanotubes not only isothermally self-assemble directly inside the microcompartments but also self-organize into dynamic higher-order structures resembling rings and dynamic networks. Our study provides an advantageous method for in situ assembly of programmable biomolecular scaffolds and materials using synthetic DNA strands without requirements of thermal treatment.

Chemistry↗
DOE OSTI2020

Biosystems Design by Machine Learning

Biosystems such as enzymes, pathways, and whole cells have been increasingly explored for biotechnological applications. Yet, the intricate connectivity and complexity of biosystems pose a major hurdle in designing biosystems with desired features. As -omics and other high throughput technologies have been rapidly developed, the promise of applying machine learning (ML) techniques in biosystems design has started to become a reality. ML models enable the identification of patterns within complicated biological data across multiple scales of analysis and can augment biosystems design applications by predicting new candidates for optimized performance. ML is being used at every stage of biosystems design to help find non-obvious engineering solutions with fewer design iterations. In this review, we first describe commonly used models and modeling paradigms within ML. We then discuss some applications of these models that have already shown success in biotechnological applications. Moreover, we discuss successful applications at all scales of biosystems design, including nucleic acids, genetic circuits, proteins, pathways, genomes, and bioprocess. Lastly, we discuss some limitations of these methods and potential solutions as well as prospects of the combination of ML and biosystems design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Discovery of protein- and DNA-imperceptible nanoparticle hard coating using gel-based reaction tuning

The seemingly inevitable protein corona appears to be an insurmountable obstacle to wider application of functional nanomaterials in biotechnology. The accumulation of serum proteins can block targeting functionalities and alter the in vivo fate of these nanomaterials. Here we demonstrate a method to generate non-stick, robustly passivated functional nanoparticles (NPs) using a tailored silica coating. We apply agarose gel electrophoresis to sensitively evaluate protein binding to NPs with different surface chemistry. Using gel banding and retardation as a read-out for protein adsorption, we optimize the surface chemistry to yield a mixed charge surface which displays remarkable binding resistance to a wide range of serum proteins and nucleic acids. Here, the hard silica shell also protects the functional NP core in harsh environments (down to pH 1) while still showing the ability to be targeted for cellular uptake with little or no non-specific binding.

36 MATERIALS SCIENCE↗
DOE OSTI2021

PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic 5 engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas 6 genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, in this paper we report 7 peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand 8 DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on 9 target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much 10 smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, 11 demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

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 OSTI2018

DNA-Encoded Protein Janus Nanoparticles

Asymmetric functionality and directional interactions, which are characteristic of noncentrosymmetric particles, such as Janus particles, present an opportunity to encode particles with properties, but also a great synthetic challenge. Here, we exploit the chemical anisotropy of proteins, and the versatile chemistry of DNA to synthesize a protein-based Janus nanoparticle comprised of two proteins encoded with sequence-specific nucleic acid domains, tethered together by an interprotein “DNA bond”. We use these novel nanoparticles to realize a new class of three-dimensional superlattice, only possible when two sides of the particle are modified with orthogonal oligonucleotide sequences. The low symmetry, intrinsic to Janus particles, enables the realization of unprecedented multicomponent nanoparticle superlattices with unique, hexagonal layered architectures. In addition, the interprotein “DNA bond” can be modulated to selectively expand the lattice in a single direction. The results presented herein not only emphasize the power of rationally designing nanoscale building blocks to create highly engineered colloidal crystals, but also establish a precedent for applications of multidomain DNA-encoded nanoparticles, especially in the field of colloidal crystallization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

The past, present and future of microbiome analyses

Microbes evolved on Earth approximately 3.5 billion years ago and eventually occupied every habitable environment in its biosphere. While microorganisms are responsible for key functions on Earth including carbon and nutrient cycling and determining the health and disease of its inhabitants (i.e. plants and animals), >99% of the estimated trillions of microbes have yet to be discovered.1 In addition, high microbial diversity has made it difficult to study specific functions carried out by complex microbial communities within "microbiomes", such as those inhabiting the soil and human gut.2,3 Fortunately, technological advances over the last few decades have greatly facilitated studies of complex microbiomes and their functions. Here we will specifically discuss advances related to nucleic acid sequencing and mass spectrometry analyses that have enabled exploration and understanding of complex microbiomes in a range of environments and our own bodies.4,5,6,7

Next Generation sequencing and Mass Spec↗
DOE OSTI2017

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Structural visualization of RNA polymerase III transcription machineries

Abstract RNA polymerase III (Pol III) transcription initiation requires the action of the transcription factor IIIB (TFIIIB) and is highly regulated. Here, we determine the structures of Pol III pre-initiation complexes (PICs) using single particle cryo-electron microscopy (cryo-EM). We observe stable Pol III–TFIIIB complexes using nucleic acid scaffolds mimicking various functional states, in which TFIIIB tightly encircles the upstream promoter DNA. There is an intricate interaction between TFIIIB and Pol III, which stabilizes the winged-helix domains of the C34 subunit of Pol III over the active site cleft. The architecture of Pol III PIC more resembles that of the Pol II PIC than the Pol I PIC. In addition, we also obtain a 3D reconstruction of Pol III in complex with TFIIIB using the elongation complex (EC) scaffold, shedding light on the mechanism of facilitated recycling of Pol III prior to transcription re-initiation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry

The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5’-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3’ flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.

59 BASIC BIOLOGICAL SCIENCES↗