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At least 253 records · Page 14

All sources NASA NTRS DOE OSTI

DOE OSTI2021

Chemical amplification based on fluid partitioning

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

Anderson, Brian L.↗

DOE OSTI2024

Single-Dose Immunogenic DNA Vaccines Coding for Live-Attenuated Alpha- and Flaviviruses

Single-dose, immunogenic DNA (iDNA) vaccines coding for whole live-attenuated viruses are reviewed. This platform, sometimes called immunization DNA, has been used for vaccine development for flavi- and alphaviruses. An iDNA vaccine uses plasmid DNA to launch live-attenuated virus vaccines in vitro or in vivo. When iDNA is injected into mammalian cells in vitro or in vivo, the RNA genome of an attenuated virus is transcribed, which starts replication of a defined, live-attenuated vaccine virus in cell culture or the cells of a vaccine recipient. In the latter case, an immune response to the live virus vaccine is elicited, which protects against the pathogenic virus. Unlike other nucleic acid vaccines, such as mRNA and standard DNA vaccines, iDNA vaccines elicit protection with a single dose, thus providing major improvement to epidemic preparedness. Still, iDNA vaccines retain the advantages of other nucleic acid vaccines. In summary, the iDNA platform combines the advantages of reverse genetics and DNA immunization with the high immunogenicity of live-attenuated vaccines, resulting in enhanced safety and immunogenicity. This vaccine platform has expanded the field of genetic DNA and RNA vaccines with a novel type of immunogenic DNA vaccines that encode entire live-attenuated viruses.

60 APPLIED LIFE SCIENCES↗

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 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 OSTI2020

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.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

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↗

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 OSTI2022

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1991

Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA

Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.

NASA Discipline Exobiology↗

NASA NTRS2012

Methods for determining the genetic affinity of microorganisms and viruses

Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

Fox, George E.↗

NASA NTRS2018

Enantiomer Excesses in Meteoritic Organic Compounds: a Role for Radiation-Magnetism?

Carbonaceous chondrites contain an insoluble “macromolecular” carbon phase as well as discrete soluble organic compounds including amino acids and sugar derivatives. Both the macromolecular carbon and the sugar derivatives are thought to have formed through aqueous formaldehyde chemistry. Formaldehyde is a ubiquitous molecule in interstellar space environments and therefore its chemistry was likely important during the formation of the Earth and other planets. It was also likely delivered throughout Earth's history: it is found in comets and (asteroidal) meteorites. Among the reactions of formaldehyde, those in water are thought to be an important part of the primordial route to today’s nucleic acids, DNA and RNA. A plausible pre-biotic path to these biopolymers would begin with the "formose" reaction, the self-reaction of formaldehyde in an alkaline aqueous medium. Formaldehyde's inclusion in the young solar system’s planets would have provided a relatively fast route to a variety of hydroxylated compounds related to nucleic acids and metabolism, including sugars, sugar acids, sugar alcohols, and formaldehyde polymers.

Cooper, G.↗

NASA NTRS1992

On the early emergence of reverse transcription: theoretical basis and experimental evidence

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.

Non-NASA Center↗

NASA NTRS1998

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

Non-NASA Center↗

DOE OSTI2024

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

60 APPLIED LIFE SCIENCES↗