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At least 361 records · Page 20
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Structure of RADX and mechanism for regulation of RAD51 nucleofilaments

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX’s mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments.

Science & Technology - Other Topics↗
DOE OSTI2023

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures

Abstract DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site‐specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to “smart” functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

Yang, Yunqi↗
DOE OSTI2023

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, in this study, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site‐specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to “smart” functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

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

Photoelectron Spectroscopy and Theoretical Investigations of Gaseous Doubly Deprotonated 2’-Deoxynucleoside 5’-Monophosphate Dianions

This work reports a systematic photoelectron spectroscopy and theoretical investigation on the electronic and geometric structures of four doubly deprotonated 2’-deoxynucleoside 5’-monophosphate dianions, the smallest quintessential DNA building block. These dianions are intrinsically stable with their adiabatic/vertical detachment energies (ADE/VDE) ranging from 0.85/1.07 (A), 1.05/1.30 (G) to 1.20/1.50 (C) and 1.80/2.10 eV (T). The repulsive Coulomb barrier against electron detachment is 2.0 eV for purines and 2.5 eV for pyrimidines. Dianions are deprotonated at the phosphate group and the amino group of a nucleobase. The calculated ADE/VDE values, as well as the simulated spectrum, agree well with the experimental results. The ?-type HOMO orbital resides on the nucleobase moiety for each dianion. This spatial distribution of HOMO suggests that the most loosely bound electron is detached along the direction perpendicular to the nucleobase. When combined with the previous results this work makes complete the depiction of basic building blocks of DNA at the molecular level.

Yuan, Qinqin↗
DOE OSTI2025

Encoding hierarchical 3D architecture through inverse design of programmable bonds

The ability to fabricate materials and devices at small scales by design has resulted in tremendous technological progress. However, the need for engineered three-dimensional (3D) nanoscale materials requires new strategies for organizing nanocomponents. Here we demonstrate an inverse design approach for the assembly of nanoparticles into hierarchically ordered 3D organizations using DNA voxels with directional, addressable bonds. By identifying intrinsic symmetries in repeating mesoscale structural motifs, we prescribe a set of voxels, termed a mesovoxel, that are assembled into target 3D crystals. The relationship between different degrees of encoded information used for voxel bonds and the fidelity of assembly is investigated using experimental and computational methods. We apply this assembly strategy to create periodic 3D nanoparticle ordered organizations, including structures with low-dimensional elements, helical motifs, a nanoscale analogue of a face-centred perovskite crystal and a distributed Bragg reflector based on a crystal with plasmonic and photonic length-scale regimes.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs

Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. Here, to understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Toward three-dimensional DNA industrial nanorobots

Nanoscale industrial robots have potential as manufacturing platforms and are capable of automatically performing repetitive tasks to handle and produce nanomaterials with consistent precision and accuracy. We demonstrate a DNA industrial nanorobot that fabricates a three-dimensional (3D), optically active chiral structure from optically inactive parts. By making use of externally controlled temperature and ultraviolet (UV) light, our programmable robot, ~100 nanometers in size, grabs different parts, positions and aligns them so that they can be welded, releases the construct, and returns to its original configuration ready for its next operation. Our robot can also self-replicate its 3D structure and functions, surpassing single-step templating (restricted to two dimensions) by using folding to access the third dimension and more degrees of freedom. Our introduction of multiple-axis precise folding and positioning as a tool/technology for nanomanufacturing will open the door to more complex and useful nano- and microdevices.

Robotics↗
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↗
DOE OSTI2023

Effect of Hydrophilicity-Imparting Substituents on Exciton Delocalization in Squaraine Dye Aggregates Covalently Templated to DNA Holliday Junctions

Molecular aggregates exhibit emergent properties, including the collective sharing of electronic excitation energy known as exciton delocalization, that can be leveraged in applications such as quantum computing, optical information processing, and light harvesting. In a previous study, we found unexpectedly large excitonic interactions (quantified by the excitonic hopping parameter J m,n ) in DNA-templated aggregates of squaraine (SQ) dyes with hydrophilic-imparting sulfo and butylsulfo substituents. Here, we characterize DNA Holliday junction (DNA-HJ) templated aggregates of an expanded set of SQs and evaluate their optical properties in the context of structural heterogeneity. Specifically, we characterized the orientation of and J m,n between dyes in dimer aggregates of non-chlorinated and chlorinated SQs. Here, three new chlorinated SQs that feature a varying number of butylsulfo substituents were synthesized and attached to a DNA-HJ via a covalent linker to form adjacent and transverse dimers. Various characteristics of the dye, including its hydrophilicity (in terms of log P o/w ) and surface area, and of the substituents, including their local bulkiness and electron withdrawing capacity, were quantified computationally.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The influence of Holliday junction sequence and dynamics on DNA crystal self-assembly

The programmable synthesis of rationally engineered crystal architectures for the precise arrangement of molecular species is a foundational goal in nanotechnology, and DNA has become one of the most prominent molecules for the construction of these materials. In particular, branched DNA junctions have been used as the central building block for the assembly of 3D lattices. Here, crystallography is used to probe the effect of all 36 immobile Holliday junction sequences on self-assembling DNA crystals. Contrary to the established paradigm in the field, most junctions yield crystals, with some enhancing the resolution or resulting in unique crystal symmetries. Unexpectedly, even the sequence adjacent to the junction has a significant effect on the crystal assemblies. Six of the immobile junction sequences are completely resistant to crystallization and thus deemed “fatal,” and molecular dynamics simulations reveal that these junctions invariably lack two discrete ion binding sites that are pivotal for crystal formation. The structures and dynamics detailed here could be used to inform future designs of both crystals and DNA nanostructures more broadly, and have potential implications for the molecular engineering of applied nanoelectronics, nanophotonics, and catalysis within the crystalline context.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The engineered single guide RNA structure as a biomarker for gene-editing reagent exposure

Abstract CRISPR arrays and CRISPR-associated (Cas) proteins comprise a prevalent adaptive immune system in bacteria and archaea. These systems defend against exogenous parasitic mobile genetic elements. The adaption of single effector CRISPR-Cas systems has massively facilitated gene-editing due to the reprogrammable guide RNA. The guide RNA affords little priming space for conventional PCR-based nucleic acid tests without foreknowledge of the spacer sequence. Further impeding detection of gene-editor exposure, these systems are derived from human microflora and pathogens ( Staphylococcus pyogenes , Streptococcus aureus , etc.) that contaminate human patient samples. The single guide RNA—formed from the CRISPR RNA (crRNA) and transactivating RNA (tracrRNA)—harbors a variable tetraloop sequence between the two RNA segments, complicating PCR assays. Identical single effector Cas proteins are used for gene-editing and naturally by bacteria. Antibodies raised against these Cas proteins are unable to distinguish CRISPR-Cas gene-editors from bacterial contaminant. To overcome the high potential for false positives, we have developed a DNA displacement assay to specifically detect gene-editors. We leveraged the single guide RNA structure as an engineered moiety for gene-editor exposure that does not cross-react with bacterial CRISPRs. Our assay has been validated for five common CRISPR systems and functions in complex sample matrices.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Robustness of Optical Response for Self-Assembled Plasmonic Metamaterials with Morphological Disorder and Surface Roughness

Bottom-up fabrication of metallized biotemplated nanostructures to form specific plasmonic nanoresonators holds promise as a means of achieving large-scale optical metamaterials. However, in contrast to top-down methods, the stochastic growth of self-assembled nanoresonators is prone to significant disorder and surface roughness, which naturally raise an important question about the robustness of their resonant properties in terms of structural imperfections. An aggregated-random-sphere model is developed to mimic the nucleated growth of metallized DNA origami assembly, leading to meta-atoms with realistic, experimentally observed morphological disorder and surface roughness. Using the well-known split-ring-resonator (SRR) motif as an example, the resonant properties of meta-atoms under different levels of roughness are investigated and a strong tolerance of optical response against morphological disorder is revealed. It is found that in SRRs, even with dramatic roughness introduced, the expected resonances are still observed, despite broadening line shapes compared to ideal smooth structure. Only for extreme disorder, which causes drastic segmentation of SRRs, does the resonant response disappear. Finally, the demonstrations are very encouraging for the prospects of bottom-up fabrication toward versatile functional metamaterials and metadevices.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Mono- and bilayer smectic liquid crystal ordering in dense solutions of “gapped” DNA duplexes

Significance Liquid crystalline behavior of DNA has been studied for decades, yet only recently has one of the most fundamental liquid crystal phases—the smectic phase in which rod-like objects assemble into stacks of fluid layers—been demonstrated in this system. Here, we describe distinct bilayer and monolayer smectic phases in solutions of “gapped” DNA (GDNA), consisting of two DNA duplexes connected by a single strand. The smectic order is amphotropic (sensitive to temperature as well as concentration), and the layer structure is stable against significant perturbations of the GDNA construct (e.g., “gap” length variation and asymmetry in duplex lengths). The temperature sensitivity may provide a means to compare end-to-end interaction strengths between DNA duplexes at biologically relevant concentrations.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Structure of a HIV-1 IN-Allosteric inhibitor complex at 2.93 Å resolution: Routes to inhibitor optimization

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy-terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we report a 2.93 Å X-ray crystal structure of the minimal ternary complex between CCD , CTD , and the ALLINI BI-224436. This structure reveals an asymmetric ternary complex with a prominent network of π-mediated interactions that suggest specific avenues for future ALLINI development and optimization.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering

Abstract Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular Regulatory Mechanisms Drive Emergent Pathogenetic Properties of Neisseria gonorrhoeae

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, with an estimated 87 million annual cases worldwide. N. gonorrhoeae predominantly colonizes the male and female genital tract (FGT). In the FGT, N. gonorrhoeae confronts fluctuating levels of nutrients and oxidative and non-oxidative antimicrobial defenses of the immune system, as well as the resident microbiome. One mechanism utilized by N. gonorrhoeae to adapt to this dynamic FGT niche is to modulate gene expression primarily through DNA-binding transcriptional regulators. Here, we describe the major N. gonorrhoeae transcriptional regulators, genes under their control, and how these regulatory processes lead to pathogenic properties of N. gonorrhoeae during natural infection. We also discuss the current knowledge of the structure, function, and diversity of the FGT microbiome and its influence on gonococcal survival and transcriptional responses orchestrated by its DNA-binding regulators. We conclude with recent multi-omics data and modeling tools and their application to FGT microbiome dynamics. Understanding the strategies utilized by N. gonorrhoeae to regulate gene expression and their impact on the emergent characteristics of this pathogen during infection has the potential to identify new effective strategies to both treat and prevent gonorrhea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CHARMM-GUI Drude prepper for molecular dynamics simulation using the classical Drude polarizable force field

Explicit treatment of electronic polarizability in empirical force fields (FFs) represents an extension over a traditional additive or pairwise FF and provides a more realistic model of the variations in electronic structure in condensed phase, macromolecular simulations. To facilitate utilization of the polarizable FF based on the classical Drude oscillator model, Drude Prepper has been developed in CHARMM-GUI. Drude Prepper ingests additive CHARMM protein structures file (PSF) and pre-equilibrated coordinates in CHARMM, PDB, or NAMD format, from which the molecular components of the system are identified. These include all residues and patches connecting those residues along with water, ions, and other solute molecules. This information is then used to construct the Drude FF-based PSF using molecular generation capabilities in CHARMM, followed by minimization and equilibration. In addition, inputs are generated for molecular dynamics (MD) simulations using CHARMM, GROMACS, NAMD, and OpenMM. Validation of the Drude Prepper protocol and inputs is performed through conversion and MD simulations of various heterogeneous systems that include proteins, nucleic acids, lipids, polysaccharides, and atomic ions using the aforementioned simulation packages. Stable simulations are obtained in all studied systems, including 5 μs simulation of ubiquitin, verifying the integrity of the generated Drude PSFs. Additionally, the ability of the Drude FF to model variations in electronic structure is shown through dipole moment analysis in selected systems. Finally, the capabilities and availability of Drude Prepper in CHARMM-GUI is anticipated to greatly facilitate the application of the Drude FF to a range of condensed phase, macromolecular systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗