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At least 415 records · Page 23

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DOE OSTI2023

Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices

Methods for the automated template-free synthesis of user-defined sequence controlled biopolymers using microfluidic devices are described. The methods facilitate simultaneous synthesis of up to thousands of uniquely addressed biopolymers from the controlled movement and combination of regents as fluid droplets using microfluidic and EWOD-based systems. In some forms, biopolymers including nucleic acids, peptides, carbohydrates, and lipids are synthesized from step-wise assembly of building blocks based on a user-defined sequence of droplet movements. In some forms, the methods synthesize uniquely addressed nucleic acids of up to 1,000 nucleotides in length. Methods for adding, removing and changing barcodes on biopolymers are also provided. Biopolymers synthesized according to the methods, and libraries and databases thereof are also described. Modified biopolymers, including chemically modified nucleotides and biopolymers conjugated to other molecules are described.

Banal, James↗

NASA NTRS2000

Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides

Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template-directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates.

Non-NASA Center↗

DOE OSTI2021

Diagnostic Testing for COVID-19 Bridging Study for QIAamp Viral RNA Extraction vs Beckman RNAdvance vs Thermofisher MagMAX

This report describes testing that was performed by LANL’s Biological Agent Testing Lab (BATL) to validate modifications to the CDC EUA 2019-Novel Coronavirus (2019- nCoV) Real-Time RT-PCR Diagnostic Panel (EUA-CDC-nCoV-IFU). BATL intends to implement the modifications to increase thoughput for daily testing . BATL validated the viral RNA extraction process, using the orignial component, QIAamp Viral RNA Mini Kit (Cat # 52906) and the new components, Beckman Coulter magnetic 96-well plate RNAdvance Viral kit (Cat # C63510), Thermofisher MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Cat # A48310). Equivalency was demonstrated between the original component and the Beckman Coulter magnetic 96-well plate RNAdvance Viral kit (Cat # C63510). Equivalency was also demonstrated between the original component and the Thermofisher MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Cat # A48310). Subsequently, substitution of the original component with either of these kits for viral RNA extraction increased BATL’s extraction capability from 100 samples per day to 279 samples per day.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Spectroscopic Chemical Sensing and Imaging: From Plants to Animals and Humans

Chemical sensing and imaging technologies are of great importance in medical diagnostics and environmental sensing due to their ability to detect and localize chemical targets and provide valuable information in real-time. Biophotonic techniques are the most promising for in vivo applications due to their minimal invasivity. Our laboratory has introduced various biophotonics-based technologies for chemical sensing and imaging for biochemical sensing, medical diagnostics, and fundamental research. Over the years, we have developed a wide variety of fluorescence and surface-enhanced Raman scattering (SERS)-based technologies for the detection of biomarkers for cancer and other diseases. This paper provides an overview of the research on chemical and biological sensors developed in our laboratory, highlighting our work on in vivo imaging and sensing, including minimally invasive detection of endogenous fluorophores associated with malignant tissue, SERS-tag localization of cancer cells and tissues, and SERS-based detection of nucleic acid biotargets and its feasibility for in vivo applications. This manuscript also presents new development on the use of Raman imaging of SERS-labeled nanoprobes incubated in leaves for use in biofuel research, laying the foundation for studies on functional imaging of nucleic acid biomarkers in plants.

Strobbia, Pietro (ORCID:0000000308846185)↗

DOE OSTI2024

Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Here, each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2015

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2018

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

Thompson, David N.↗

DOE OSTI2020

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2014

Local dynamics of proteins and DNA evaluated from crystallographic B factors

Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are surprising. The features discriminating different types of residues are less pronounced in structures with lower crystallographic resolution. Some of the observed trends are likely to be the consequence of improper refinement protocols that may need to be rectified.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2021

Cellular mRNA triggers structural transformation of Ebola virus matrix protein VP40 to its essential regulatory form

The Ebola virus matrix protein VP40 forms distinct structures linked to distinct functions in the virus life cycle. Dimeric VP40 is a structural protein associated with virus assembly, while octameric, ring-shaped VP40 is associated with transcriptional control. In this study, we show that suitable nucleic acid is sufficient to trigger a dynamic transformation of VP40 dimer into the octameric ring. Deep sequencing reveals a binding preference of the VP40 ring for the 3' untranslated region of cellular mRNA and a guanine- and adenine-rich binding motif. Complementary analyses of the nucleic-acid-induced VP40 ring by native mass spectrometry, electron microscopy, and X-ray crystal structures at 1.8 and 1.4 Å resolution reveal the stoichiometry of RNA binding, as well as an interface involving a key guanine nucleotide. The host factor-induced structural transformation of protein structure in response to specific RNA triggers in the Ebola virus life cycle presents unique opportunities for therapeutic inhibition.

3′ UTR↗

DOE OSTI2020

Efficacy and Delivery of Novel FAST Agents for Coronaviruses

We proposed to test and develop advanced delivery for novel agents from our collaborators Facile Accelerated Specific Therapeutics (FAST) platform to reduce coronavirus replication. Sachi Bioworks Inc., Prof. Anushree Chatterjee, and Prof. Prashant Nagpal at the University of Colorado Boulder have developed a bioinformatics and synthesis pipeline to produce sequence specific theranostic agents (agents that can be therapies and/or diagnostics) that are inherently transported into the cytoplasm of mammalian host cells and sequence-specifically interfere in nucleic acid replication. The agent comprises a small nanoparticle (2-5 nm) chosen for ideal cellular transport and/or imaging conjugated to a short, synthetic DNA analog oligomer designed for binding to one or more target viral sequences. The sequence specific binding of the FAST agent to its target prevents nucleic acid replication due to its high affinity binding. While the small nanoparticle facilitates delivery in vitro, we plan to package the FAST agents into a larger nanoparticle (80-300 nm) for future in vivo delivery applications. Our team at Sandia has expertise encapsulating biomolecules including protein, DNA, and RNA into solid lipid nanoparticles (LNP) and lipid coated mesoporous silica nanoparticles (LC-MSN) and shown successful delivery in mouse models to multiple tissues. Our team focused on formulation parameters for FAST agents into lipid nanoparticles (LNP) and lipid coated mesoporous silica nanoparticles (LC-MSN) for enhanced delivery and/or efficacy and in vivo translation. We used lipid formulas that have been shown in literature to facility in vitro and more importantly, in vivo delivery. In our work discussed below, we successfully demonstrate loading and release of FAST agents on silica core and stable LC-MSN in a reasonable size range for in vivo testing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Structural basis for TNA synthesis by an engineered TNA polymerase

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(L)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants.

Science & Technology - Other Topics↗

DOE OSTI2018

Colorimetric-Luminance Readout for Quantitative Analysis of Fluorescence Signals with a Smartphone CMOS Sensor

Smartphones have shown promise as an enabling technology for portable and distributed point-of-care diagnostic tests. The CMOS camera sensor can be used for detecting optical signals, including fluorescence for applications such as isothermal nucleic acid amplification tests. However, such analysis is typically limited mostly to end point detection of single targets. In this paper, we present a smartphone-based image analysis pipeline that utilizes the CIE xyY (chromaticity-luminance) color space to measure the luminance (in lieu of RGB intensities) of fluorescent signals arising from nucleic acid amplification targets, with a discrimination sensitivity (ratio between the positive to negative signals), which is an order of magnitude more than traditional RGB intensity based analysis. Furthermore, the chromaticity part of the analysis enables reliable multiplexed detection of different targets labeled with spectrally separated fluorophores. We apply this chromaticity-luminance formulation to simultaneously detect Zika and chikungunya viral RNA via end point RT-LAMP (Reverse transcription Loop-Mediated isothermal amplification). We also show real time LAMP detection of Neisseria gonorrhoeae samples down to a copy number of 3.5 copies per 10 μL of reaction volume in our smartphone-operated portable LAMP box. Finally, our chromaticity-luminance analysis is readily adaptable to other types of multiplexed fluorescence measurements using a smartphone camera.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRSDate not supplied

Possible Mechanism(s) in the Photo-Magnetic Production of Enantiomer Excesses.

Among the multitude of organic compounds identified in carbonaceous chondrites are sugar acids ((HO-R)xCOOH), i.e., oxidized counterparts of the common aldehyde sugars (e.g., glyceraldehyde, erythrose, ribose, glucose, etc.). In the historical study of extraterrestrial compounds chiral analyses are particularly significant in origins-of-life studies. Chiral compounds are composed of two non-superposable mirror images or "enantiomers", analogous to a left and right hand. Such molecules are important in contemporary life because biological polymers (proteins, nucleic acids, etc.) are homochiral, i.e., their chiral monomers usually consist of only one of the two enantiomers. It has been shown, at least in one lab setting, that abiotic syntheses of significantly sized RNA polymers do not proceed if the monomers are present in racemic (50:50) amounts. The latter results may argue for, at least, significant enantiomer excesses in organic compounds before the origin of nucleic acids and life. Therefore, the finding that amino acids and sugar acids in carbonaceous chondrites possess enantiomeric excesses may aid in understanding one source of precursors to homochirality. Note: Attachment contains full abstract.

G. Cooper↗

NASA NTRS1994

Double layer hydroxide minerals as host structures for bioorganic molecules

A central problem in molecular evolution concerns the selective concentration from dilute, multicomponent solution of source molecules into a reactive environment, where formation of larger molecular assemblages can take place. Minerals consisting of positively charged, separable metal hydroxide sheets have proven capable of these functions. This common structural type is represented by minerals such as pyroaurite (Mg-Fe(3+) hydroxide), hydrotalcite (Mg-Al), green rust Fe(2+)-Fe(3+) and others. Effective interlayer sorption is demonstrated for orthophosphate and condensed phosphates, anionic alkyl compounds, polypeptides, nucleic acids, cyanide complexes and glycolaldehyde phosphate, the latter shown to readily oligomerize to form and selectively retain racemic hexose -2, 4, 6-phosphates, preferentially of altrose (Pitsch, et al, 1993). The selective aldomerization and retention effects correlate with the charge distribution in the host mineral structure and the stereochemistry of the substrate molecules. Interaction between nucleic acid bases, and between the cyanide groups of glycolaldehyde phosphate nitrile at the low water activity in the mineral interlayer is indicated by doubling of the monomeric separation of the hydroxide mineral sheets.

Lee, Ton↗

DOE OSTI2019

Mosaic vaccines for serotype a foot-and-mouth disease virus

Synthetic foot-and-mouth disease virus (FMDV) mosaic polypeptides, and nucleic acid molecules encoding the mosaic polypeptides, are described. The mosaic polypeptides have greater T-cell epitope coverage than naturally occurring FMDV polypeptides, and include common FMDV epitopes, but exclude rare FMDV epitopes. When included as part of an FMDV genome, the mosaic polypeptides permit virus replication and assembly into FMDV particles. The mosaic polypeptide and nucleic acid compositions can be used to elicit immune responses that provide protection against a broad range of serotype A FMDV strains.

60 APPLIED LIFE SCIENCES↗