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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Ultrastrong colloidal crystal metamaterials engineered with DNA

Lattice-based constructs, often made by additive manufacturing, are attractive for many applications. Typically, such constructs are made from microscale or larger elements; however, smaller nanoscale components can lead to more unusual properties, including greater strength, lighter weight, and unprecedented resiliencies. Here, solid and hollow nanoparticles (nanoframes and nanocages; frame size: ~15 nanometers) were assembled into colloidal crystals using DNA, and their mechanical strengths were studied. Nanosolid, nanocage, and nanoframe lattices with identical crystal symmetries exhibit markedly different specific stiffnesses and strengths. Unexpectedly, the nanoframe lattice is approximately six times stronger than the nanosolid lattice. Nanomechanical experiments, electron microscopy, and finite element analysis show that this property results from the buckling, densification, and size-dependent strain hardening of nanoframe lattices. Last, these unusual open architectures show that lattices with structural elements as small as 15 nanometers can retain a high degree of strength, and as such, they represent target components for making and exploring a variety of miniaturized devices.

42 ENGINEERING↗
NASA NTRS1997

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

NASA Discipline Exobiology↗
DOE OSTI2023

Allosteric autoregulation of DNA binding via a DNA-mimicking protein domain: a biophysical study of ZNF410–DNA interaction using small angle X-ray scattering

ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Coarse-Grained Simulations for the Characterization and Optimization of Hybrid Protein–DNA Nanostructures

We present here the combination of experimental and computational modeling tools for the design and characterization of protein–DNA hybrid nanostructures. Our work incorporates several features in the design of these nanostructures: (1) modeling of the protein–DNA linker identity and length; (2) optimizing the design of protein–DNA cages to account for mechanical stresses; (3) probing the incorporation efficiency of protein–DNA conjugates into DNA nanostructures. The modeling tools were experimentally validated using structural characterization methods like cryo-TEM and AFM. Our method can be used for fitting low-resolution electron density maps when structural insights cannot be deciphered from experiments, as well as enable in-silico validation of nanostructured systems before their experimental realization. Finally, these tools will facilitate the design of complex hybrid protein–DNA nanostructures that seamlessly integrate the two different biomolecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

DNA Crystals as a Template for Patterned Functional Materials

DNA nanotechnology offers a wide toolkit of molecular functionalities and scales, including intricate motifs less than 10 nm and periodic structures exceeding 100 µm. At larger scales, however, there are often significant tradeoffs for DNA structures, namely stability and mechanical strength. This work describes the design, synthesis, and characterization of a functionalized DNA crystal. Using a ligated DNA crystal grants significant freedom for various functional materials to be applied, in this case, semiconducting cadmium sulfide and palladium metal. Properties investigated in this study include stability, mechanical strength, and optoelectronic properties such as photoluminescence (PL) and electric conductivity. Significant changes are observed based on the functional material applied to DNA crystals. The Young's modulus of the crystal varies by about five orders of magnitude when functionalized with palladium. PL and semiconductive behaviors were observed when cadmium sulfide was attached. These crystals represent an expansion of the capabilities of DNA structures at these length scales, and additionally a platform for future studies exchanging the materials or altering the ligation scheme.

CdS↗
DOE OSTI2023

Structure of the WYL-domain containing transcription activator, DriD, in complex with ssDNA effector and DNA target site

Transcription regulators play central roles in orchestrating responses to changing environmental conditions. Recently the Caulobacter crescentus transcription activator DriD, which belongs to the newly defined WYL-domain family, was shown to regulate DNA damage responses independent of the canonical SOS pathway. However, the molecular mechanisms by which DriD and other WYL-regulators sense environmental signals and recognize DNA are not well understood. We showed DriD DNA-binding is triggered by its interaction with ssDNA, which is produced during DNA damage. Here we describe the structure of the full-length C. crescentus DriD bound to both target DNA and effector ssDNA. DriD consists of an N-terminal winged-HTH (wHTH) domain, linker region, three-helix bundle, WYL-domain and C-terminal WCX-dimer domain. Strikingly, DriD binds DNA using a novel, asymmetric DNA-binding mechanism that results from different conformations adopted by the linker. Although the linker does not touch DNA, our data show that contacts it makes with the wHTH are key for specific DNA binding. The structure indicates how ssDNA-effector binding to the WYL-domain impacts wHTH DNA binding. In conclusion, we present the first structure of a WYL-activator bound to both effector and target DNA. The structure unveils a unique, asymmetric DNA binding mode that is likely conserved among WYL-activators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Control of transcription elongation and DNA repair by alarmone ppGpp

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Targeting the Y-box Binding Protein-1 Axis to Overcome Radiochemotherapy Resistance in Solid Tumors

Multifunctional Y-box binding protein-1 (YB-1) is highly expressed in different human solid tumors and is involved in various cellular processes. DNA damage is the major mechanism by which radiochemotherapy (RCT) induces cell death. On induction of DNA damage, a multicomponent signal transduction network, known as the DNA damage response, is activated to induce cell cycle arrest and initiate DNA repair, which protects cells against damage. YB-1 regulates nearly all cancer hallmarks described to date by participating in DNA damage response, gene transcription, mRNA splicing, translation, and tumor stemness. YB-1 lacks kinase activity, and p90 ribosomal S6 kinase and AKT are the key kinases within the RAS/mitogen-activated protein kinase and phosphoinositide 3-kinase pathways that directly activate YB-1. Thus, the molecular targeting of ribosomal S6 kinase and AKT is thought to be the most effective strategy for blocking the cellular function of YB-1 in human solid tumors. In this review, after describing the prosurvival effect of YB-1 with a focus on DNA damage repair and cancer cell stemness, clinical evidence will be provided indicating an inverse correlation between YB-1 expression and the treatment outcome of solid tumors after RCT. In the interest of being concise, YB-1 signaling cascades will be briefly discussed and the current literature on YB-1 posttranslational modifications will be summarized. Finally, the current status of targeting the YB-1 axis, especially in combination with RCT, will be highlighted.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2023

Hints of Growth Mechanism Left in Supercrystals

Supercrystals of DNA-functionalized nanoparticles arevisualizedin three dimensions using X-ray ptychographic tomography, and theirreciprocal spaces are mapped with small-angle X-ray scattering inorder to better understand their internal defect structures. X-rayptychographic tomography reveals various types of defects in an assemblythat otherwise exhibits a single crystalline diffraction pattern.On average, supercrystals composed of smaller nanoparticles are smallerin size than supercrystals composed of larger particles. Additionally,supercrystals composed of small nanoparticles are typically aggregatedinto larger "necklace-like" structures. Within theselarger structures, some but not all pairs of connected domains arecoherent in their relative orientations. In contrast, supercrystalscomposed of larger nanoparticles with longer DNA ligands typicallyform faceted crystals. The combination of these two complementaryX-ray techniques reveals that the crystalline assemblies grow by aggregationof smaller assemblies followed by rearrangement of nanoparticles.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2017

Dependence of Early and Late Chromosomal Aberrations on Radiation Quality and Cell Types

Exposure to radiation induces different types of DNA damage, increases mutation and chromosome aberration rates, and increases cellular transformation in vitro and in vivo. The susceptibility of cells to radiation depends on genetic background and growth condition of cells, as well as types of radiation. Mammalian cells of different tissue types and with different genetic background are known to have different survival rate and different mutation rate after cytogenetic insults. Genomic instability, induced by various genetic, metabolic, and environmental factors including radiation, is the driving force of tumorigenesis. Accurate measurements of the relative biological effectiveness (RBE) is important for estimating radiation-related risks. To further understand genomic instability induced by charged particles and their RBE, we exposed human lymphocytes ex vivo, human fibroblast AG1522, human mammary epithelial cells (CH184B5F5/M10), and bone marrow cells isolated from CBA/CaH(CBA) and C57BL/6 (C57) mice to high energy protons and Fe ions. Normal human fibroblasts AG1522 have apparently normal DNA damage response and repair mechanisms, while mammary epithelial cells (M10) are deficient in the repair of DNA DSBs. Mouse strain CBA is radio-sensitive while C57 is radio-resistant. Metaphase chromosomes at different cell divisions after radiation exposure were collected and chromosome aberrations were analyzed as RBE for different cell lines exposed to different radiations at various time points up to one month post irradiation.

Lu, Tao↗
DOE OSTI2021

DNA origami single crystals with Wulff shapes

DNA origami technology has proven to be an excellent tool for precisely manipulating molecules and colloidal elements in a three-dimensional manner. However, fabrication of single crystals with well-defined facets from highly programmable, complex DNA origami units is a great challenge. Here, we report the successful fabrication of DNA origami single crystals with Wulff shapes and high yield. By regulating the symmetries and binding modes of the DNA origami building blocks, the crystalline shapes can be designed and well-controlled. The single crystals are then used to induce precise growth of an ultrathin layer of silica on the edges, resulting in mechanically reinforced silica-DNA hybrid structures that preserve the details of the single crystals without distortion. The silica-infused microcrystals can be directly observed in the dry state, which allows meticulous analysis of the crystal facets and tomographic 3D reconstruction of the single crystals by high-resolution electron microscopy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI

Abstract Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Construction of Reconfigurable and Polymorphic DNA Origami Assemblies with Coiled‐Coil Patches and Patterns

Abstract DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence‐specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well‐defined micron‐scale structures through hierarchical self‐assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structures and properties. Here the integration of DNA origami hinge nanodevices and coiled‐coil peptides are reported into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, it is made multiple higher‐order assemblies (i.e., polymorphic assembly) by organizing clusters of peptides into patches or arranging single peptides into patterns on the surfaces of DNA origami to control the relative orientation of devices. The coiled‐coil interactions are used to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA‐based reconfiguration. Reconfiguration of linear assemblies leads to micron scale motions and ≈2.5‐10‐fold increase in bending stiffness. The results provide a foundation for stimulus‐responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas

Abstract Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

Neupert, Juliane↗
DOE OSTI2020

Getting to the Root of Plant–Mediated Methane Emissions and Oxidation in a Thermokarst Bog

Vascular plants are important in the wetland methane cycle, but their effect on production, oxidation, and transport has high uncertainty, limiting our ability to predict emissions. In a permafrost–thaw bog in Interior Alaska, we used plant manipulation treatments, field–deployed planar optical oxygen sensors, direct measurements of methane oxidation, and microbial DNA analyses to disentangle mechanisms by which vascular vegetation affect methane emissions. Vegetation operated on top of baseline methane emissions, which varied with proximity to the thawing permafrost margin. Emissions from vegetated plots increased over the season, resulting in cumulative seasonal methane emissions that were 4.1–5.2 g m –2 season –1 greater than unvegetated plots. Mass balance calculations signify these greater emissions were due to increased methane production (3.0–3.5 g m –2 season –1 ) and decreased methane oxidation (1.1–1.6 g m –2 season –1 ). Minimal oxidation occurred along the plant–transport pathway, and oxidation was suppressed outside the plant pathway. Our data indicate suppression of methane oxidation was stimulated by root exudates fueling competition among microbes for electron acceptors. This contention is supported by the fact that methane oxidation and relative abundance of methanotrophs decreased over the season in the presence of vegetation, but methane oxidation remained steady in unvegetated treatments; oxygen was not detected around plant roots but was detected around silicone tubes mimicking aerenchyma; and oxygen injection experiments suggested that oxygen consumption was faster in the presence of vascular vegetation. In conclusion, root exudates are known to fuel methane production, and our work provides evidence they also decrease methane oxidation.

58 GEOSCIENCES↗
DOE OSTI2023

Structure of a 10-23 deoxyribozyme exhibiting a homodimer conformation

Deoxyribozymes (DNAzymes) are in vitro evolved DNA sequences capable of catalyzing chemical reactions. The RNA-cleaving 10-23 DNAzyme was the first DNAzyme to be evolved and possesses clinical and biotechnical applications as a biosensor and a knockdown agent. DNAzymes do not require the recruitment of other components to cleave RNA and can turnover, thus they have a distinct advantage over other knockdown methods (siRNA, CRISPR, morpholinos). Despite this, a lack of structural and mechanistic information has hindered the optimization and application of the 10-23 DNAzyme. Here, we report a 2.7 Å crystal structure of the RNA-cleaving 10-23 DNAzyme in a homodimer conformation. Although proper coordination of the DNAzyme to substrate is observed along with intriguing patterns of bound magnesium ions, the dimer conformation likely does not capture the true catalytic form of the 10-23 DNAzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9 s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

59 BASIC BIOLOGICAL SCIENCES↗