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At least 397 records · Page 22

All sources NASA NTRS DOE OSTI

DOE OSTI2023

Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA

The interbacterial deaminase toxin DddA catalyzes cytosine-to-uracil conversion in double-stranded (ds) DNA and enables CRISPR-free mitochondrial base editing, but the molecular mechanisms underlying its unique substrate selectivity have remained elusive. Here, we report crystal structures of DddA bound to a dsDNA substrate containing the 5'-TC target motif. These structures show that DddA binds to the minor groove of a sharply bent dsDNA and engages the target cytosine extruded from the double helix. DddA Phe1375 intercalates in dsDNA and displaces the 5' (-1) thymine, which in turn replaces the target (0) cytosine and forms a noncanonical T–G base pair with the juxtaposed guanine. This tandem displacement mechanism allows DddA to locate a target cytosine without flipping it into the active site. Biochemical experiments demonstrate that DNA base mismatches enhance the DddA deaminase activity and relax its sequence selectivity. On the basis of the structural information, we further identified DddA mutants that exhibit attenuated activity or altered substrate preference. Our studies may help design new tools useful in genome editing or other applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

Next-Generation Sequencing Data from a CUT&RUN Study of R. toruloides IFO0880 Cse4 and Orc1 Binding Sites

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

Genome Engineering↗

DOE OSTI2021

Subsampling and DNA pooling can increase gains through genomic selection in switchgrass

Genomic selection (GS) can accelerate breeding cycles in perennial crops such as the bioenergy grass switchgrass (Panicum virgatum L.). The sequencing costs of GS can be reduced by pooling DNA samples in the training population (TP), only sequencing TP phenotypic outliers, or pooling candidate population (CP) samples. These strategies were simulated for two traits (spring vigor and anthesis date) in three breeding populations. Sequencing only the outlier 50% of the TP phenotype distribution resulted in a penalty of <5% of the predictive ability, measured using cross-validation. Predictive ability also decreased when sequencing progressively fewer TP DNA pools, but TPs constructed from only two phenotypically contrasting DNA samples retained a mean of >80% predictive ability relative to individual TP sequencing. Novel group testing methods allowed greater than one CP individual to be screened per sequenced DNA sample but resulted in a predictive ability penalty. To determine the impact of reduced sequencing, genetic gain was calculated for seven GS scenarios with variable sequencing budgets. Reduced TP sequencing and most CP pooling methods were superior to individual sequence-based GS when sequencing resources were restricted (2,000 DNA samples per 5-yr cycle). Only one scenario was superior to individual sequencing when sequencing budgets were large (8,000 DNA samples per 5-yr cycle). This study highlights multiple routes for reduced sequencing costs in GS.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1995

Mutational analysis of photosystem I polypeptides in the cyanobacterium Synechocystis sp. PCC 6803. Targeted inactivation of psaI reveals the function of psaI in the structural organization of psaL

We cloned, characterized, and inactivated the psaI gene encoding a 4-kDa hydrophobic subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. The psaI gene is located 90 base pairs downstream from psaL, and is transcribed on 0.94- and 0.32-kilobase transcripts. To identify the function of PsaI, we generated a cyanobacterial strain in which psaI has been interrupted by a gene for chloramphenicol resistance. The wild-type and the mutant cells showed comparable rates of photoautotrophic growth at 25 degrees C. However, the mutant cells grew slower and contained less chlorophyll than the wild-type cells, when grown at 40 degrees C. The PsaI-less membranes from cells grown at either temperature showed a small decrease in NADP+ photoreduction rate when compared to the wild-type membranes. Inactivation of psaI led to an 80% decrease in the PsaL level in the photosynthetic membranes and to a complete loss of PsaL in the purified photosystem I preparations, but had little effect on the accumulation of other photosystem I subunits. Upon solubilization with nonionic detergents, photosystem I trimers could be obtained from the wild-type, but not from the PsaI-less membranes. The PsaI-less photosystem I monomers did not contain detectable levels of PsaL. Therefore, a structural interaction between PsaL and PsaI may stabilize the association of PsaL with the photosystem I core. PsaL in the wild-type and PsaI-less membranes showed equal resistance to removal by chaotropic agents. However, PsaL in the PsaI-less strain exhibited an increased susceptibility to proteolysis. From these data, we conclude that PsaI has a crucial role in aiding normal structural organization of PsaL within the photosystem I complex and the absence of PsaI alters PsaL organization, leading to a small, but physiologically significant, defect in photosystem I function.

NASA Discipline Cell Biology↗

DOE OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

Genomic DNA from each sample was prepared using the Wizard Genomic DNA Purification Kit (Promega) and after, DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was used to select ~200 bp fragments and the fragments then underwent a splinkerette PCR using a Tn5-enriching forward primer and custom reverse primers for multiplexing. A final bead-based size selection was used to select for the correct length DNA. DNA was sequenced at the University of Michigan Advanced Genomics Core using Illumina sequencing with a custom read primer reading the last 10 nt of the transposon. PhiX174 DNA spike was added to the run to ensure sufficient sequence diversity on the flow cell. Then, a custom index read primer and standard Illumina primer were used to sequence the index reads and PhiX174, respectively.

Keck, James L.↗

DOE OSTI2020

Probing the Consequences of Cubic Particle Shape and Applied Field on Colloidal Crystal Engineering with DNA

Abstract In a magnetic field, cubic Fe 3 O 4 nanoparticles exhibit assembly behavior that is a consequence of a competition between magnetic dipole–dipole and ligand interactions. In most cases, the interactions between short hydrophobic ligands dominate and dictate assembly outcome. To better tune the face‐to‐face interactions, cubic Fe 3 O 4 nanoparticles were functionalized with DNA. Their assembly behaviors were investigated both with and without an applied magnetic field. Upon application of a field, the tilted orientation of cubes, enabled by the flexible DNA ligand shell, led to an unexpected crystallographic alignment of the entire superlattice, as opposed to just the individual particles, along the field direction as revealed by small and wide‐angle X‐ray scattering. This observation is dependent upon DNA length and sequence and cube dimensions. Taken together, these studies show how combining physical and chemical control can expand the possibilities of crystal engineering with DNA.

Urbach, Zachary J.↗

DOE OSTI2020

Probing the Consequences of Cubic Particle Shape and Applied Field on Colloidal Crystal Engineering with DNA

In a magnetic field, cubic Fe 3 O 4 nanoparticles exhibit assembly behavior that is a consequence of a competition between magnetic dipole-dipole and ligand interactions. In most cases, the interactions between short hydrophobic ligands dominate and dictate assembly outcome. To better tune the face-to-face interactions, cubic Fe3O4 nanoparticles were functionalized with DNA. Their assembly behaviors were investigated both with and without an applied magnetic field. Upon application of a field, the tilted orientation of cubes, enabled by the flexible DNA ligand shell, led to an unexpected crystallographic alignment of the entire superlattice, as opposed to just the individual particles, along the field direction as revealed by small and wide-angle X-ray scattering. This observation is dependent upon DNA length and sequence and cube dimensions. Taken together, these studies show how combining physical and chemical control can expand the possibilities of crystal engineering with DNA.

36 MATERIALS SCIENCE↗

DOE OSTI2025

GMFOLD: Subgraph matching for high-throughput DNA-aptamer secondary structure classification and machine learning interpretability

Aptamers are oligonucleotide receptors that bind to their targets with high affinity. Here, we consider aptamers comprised of single-stranded DNA that undergo target-binding-induced conformational changes, giving rise to unique secondary and tertiary structures. Given a specific aptamer primary sequence, there are well-established computational tools (notably mfold) to predict the secondary structure via free energy minimization algorithms. While mfold generates secondary structures for individual sequences, there is a need for a high-throughput process whereby thousands of DNA structures can be predicted in real-time for use in an interactive setting, when combined with aptamer selections that generate candidate pools that are too large to be experimentally interrogated. We developed a new Python code for high-throughput aptamer secondary structure determination (GMfold). GMfold uses subgraph matching methods to group aptamer candidates by secondary structure similarities. We also improve an open-source code, SeqFold, to incorporate subgraph matching concepts. We represent each secondary structure as a lowest-energy bipartite subgraph matching of the DNA graph to itself. These new tools enable thousands of DNA sequences to be compared based on their secondary structures, using machine-learning algorithms. This process is advantageous when analyzing sequences that arise from aptamer selections via systematic evolution of ligands by exponential enrichment (SELEX). This work is a building block for future machine-learning-informed DNA-aptamer selection processes to identify aptamers with improved target affinity and selectivity and advance aptamer biosensors and therapeutics.

Aptamer↗

DOE OSTI2020

In vitro recombination method

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

In vitro recombination method

Young, Lei↗

NASA NTRS1998

Acid-growth response and alpha-expansins in suspension cultures of bright yellow 2 tobacco

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of alpha-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple alpha-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Non-NASA Center↗

DOE OSTI2026

SEGUID v2: Extending SEGUID checksums for circular, linear, single- and double-stranded biological sequences

Background Synthetic biology involves combining different DNA fragments, each containing functional biological parts, to address specific problems. Fundamental gene-function research often requires cloning and propagating DNA fragments, such as those from the iGEM Parts Registry or Addgene, typically distributed as circular plasmids. Addgene’s repository alone offers around 150,000 plasmids. To ensure data integrity, cryptographic checksums can be calculated for the sequences. Each sequence has a unique checksum, making checksums useful for validation and quick lookups of associated annotations. For example, the SEGUID checksum uniquely identifies protein sequences with a 27-character string. Objectives The original SEGUID, while effective for protein sequences and single-stranded DNA (ssDNA), is not suitable for circular DNA since there is no natural starting position nor for double-stranded DNA (dsDNA) since two separate sequences are present. Challenges include how to uniquely represent linear dsDNA, circular ssDNA, and circular dsDNA. To meet these needs, we propose SEGUID v2, which extends the original SEGUID to handle additional types of sequences. Conclusions SEGUID v2 produces orientation and rotation invariant checksums for single-stranded, double-stranded, possibly staggered, linear, and circular DNA and RNA sequences. Customizable alphabets allow for other types of sequences. In contrast to the original SEGUID, which uses Base64, SEGUID v2 uses Base64url to encode the SHA-1 hash. This ensures SEGUID v2 checksums can be used as-is in filenames, regardless of platform, and in URLs, with minimal friction. Availability SEGUID v2 is readily available for major programming languages, distributed under the MIT license. JavaScript package seguid is available on npm, Python package seguid on PyPi, R package seguid on CRAN, and a Tcl script on GitHub. These tools, along with documentation, examples, and an online SEGUID Calculator , can be found at https://www.seguid.org .

Pereira, Humberto↗

NASA NTRS1999

Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacterium from an oil brine

Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA. The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2. The strains were mesophilic and grew at a pH range of 6-8. Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin. Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains. Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition. Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium. The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium. It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

NASA Discipline Exobiology↗

DOE OSTI2020

DelPlasmid: Finding Plasmids with Deep Learning and Machine Learning v1

Extrachromosomal genetic elements or plasmids are an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from host cell chromosomes and their frequent self-transfer. In recent years, the Department of Energy has recognized the importance of plasmid DNA to the viability and success of microbial communities and JGI has responded with funding to support the research and development of new methods and tools for the efficient sequencing of plasmid DNA. LBNL #2019-037 is responsive to this call.

Andreopoulos, William↗

DOE OSTI2023

Episomal DNA vectors for plant genetic engineering

This disclosure pertains to a novel platform for genetic engineering of chloroplasts. The disclosure provides episomal DNA vectors containing a chloroplast origin of replication. These vectors remain extra-plastomic and sustainably and autonomously replicate in chloroplasts of the plant cells transformed with the vectors and in the plants regenerated from the transformed plant cells. The episomal DNA vectors do not contain any sequence that shares sequence homology with the plastome DNA and, thus, do not get integrated into the plastome DNA. The vectors can also comprise one or more genes of interest that confer desirable characteristics to the transformed plant cells. The disclosure also provides methods of transforming plant cells with the episomal DNA vectors and regenerating from the transformed plant cells plants having desirable characteristics. The vectors and methods disclosed herein provide a significant advancement in speed, flexibility, and prospects of introducing genes into plant cells for effective metabolic engineering.

Lenaghan, Scott C.↗

DOE OSTI2021

Modeling temporal and hormonal regulation of plant transcriptional response to wounding

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2001

Ecological physiology of Synechococcus sp. strain SH-94-5, a naturally occurring cyanobacterium deficient in nitrate assimilation

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.

Nitrates/metabolism↗