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Scientific Reports May 2023The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light...
The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53's C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53's C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).
Topics: Humans; Tumor Suppressor Protein p53; Computer Simulation; Intrinsically Disordered Proteins; DNA; Mass Spectrometry; Protein Binding
PubMed: 37231156
DOI: 10.1038/s41598-023-35437-5 -
Nano Letters Sep 2019DNA nanotechnology allows for the realization of complex nanoarchitectures in which the spatial arrangements of different constituents and most functions can be enabled...
DNA nanotechnology allows for the realization of complex nanoarchitectures in which the spatial arrangements of different constituents and most functions can be enabled by DNA. When optically active components are integrated in such systems, the resulting nanoarchitectures not only provide great insights into the self-assembly of nanoscale elements in a systematic way but also impart tailored optical functionality to DNA origami. In this Letter, we demonstrate DNA-assembled multilayer nanosystems, which can carry out coordinated and reversible sliding motion powered by DNA fuels. Gold nanoparticles cross-link DNA origami filaments to define the configurations of the multilayer nanoarchitectures as well as to mediate relative sliding between the neighboring origami filaments. Meanwhile, the gold nanoparticles serve as optical probes to dynamically interact with the fluorophores tethered on the filaments, rendering in situ detection of the stepwise sliding processes possible. This work seeds the basis to implement DNA-assembled complex optical nanoarchitectures with programmability and addressability, advancing the field with new momentum.
Topics: DNA; Gold; Metal Nanoparticles
PubMed: 31438681
DOI: 10.1021/acs.nanolett.9b02565 -
Nature Aug 2022Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a...
Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.
Topics: CRISPR-Cas Systems; DNA; Gene Editing; Gene Expression; Genome; Information Storage and Retrieval; Integrases; Prokaryotic Cells; RNA; Reverse Transcription; Time Factors
PubMed: 35896746
DOI: 10.1038/s41586-022-04994-6 -
Plant Physiology Oct 2022Scientists have developed many approaches based on PCR or next-generation sequencing to localize and characterize integrated T-DNAs in transgenic plants generated by...
Scientists have developed many approaches based on PCR or next-generation sequencing to localize and characterize integrated T-DNAs in transgenic plants generated by Agrobacterium tumefaciens-mediated T-DNA transfer. However, none of these methods has the robust ability to handle all transgenic plants with diversified T-DNA patterns. Utilizing the valuable information in the whole-genome sequencing data of transgenic plants, we have developed a comprehensive approach (T-LOC) to localize and characterize T-DNA integration sites (TISs). We evaluated the performance of T-LOC on genome sequencing data from 48 transgenic rice (Oryza sativa) plants that provide real and unbiased resources of T-DNA integration patterns. T-LOC discovered 75 full TISs and reported a diversified pattern of T-DNA integration: the ideal single-copy T-DNA between two borders, multiple-copy of T-DNAs in tandem or inverted repeats, truncated partial T-DNAs with or without the selection hygromycin gene, the inclusion of T-DNA backbone, the integration at the genome repeat region, and the concatenation of multiple ideal or partial T-DNAs. In addition, we reported that DNA fragments from the two A. tumefaciens plasmids can be fused with T-DNA and integrated into the plant genome. Besides, T-LOC characterizes the genomic changes at TISs, including deletion, duplication, accurate repair, and chromosomal rearrangement. Moreover, we validated the robustness of T-LOC using PCR, Sanger sequencing, and Nanopore sequencing. In summary, T-LOC is a robust approach to studying the TISs independent of the integration pattern and can recover all types of TISs in transgenic plants.
Topics: Transformation, Genetic; DNA, Bacterial; Plants, Genetically Modified; Agrobacterium tumefaciens; Oryza
PubMed: 35640125
DOI: 10.1093/plphys/kiac225 -
The Plant Journal : For Cell and... Jan 2022Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes...
Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.
Topics: Agrobacterium tumefaciens; Arabidopsis; DNA, Bacterial; DNA, Plant; DNA-Directed DNA Polymerase; Gene Targeting; Homologous Recombination; Plants, Genetically Modified; Transgenes
PubMed: 34713516
DOI: 10.1111/tpj.15557 -
Nucleic Acids Research Sep 2023i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate....
i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate. We performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped iMs in two human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs. By simultaneously mapping G-quadruplexes (G4s), which form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.
Topics: Humans; Gene Expression Regulation; G-Quadruplexes; Regulatory Sequences, Nucleic Acid; DNA; Genomics
PubMed: 37528048
DOI: 10.1093/nar/gkad626 -
ACS Nano Jan 2023An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges...
An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges of DNA origami tiles we control their hierarchical assembly via fluorous-directed recognition. When we combine this approach with Watson-Crick base-pairing we form discrete dimeric constructs in significantly higher yield (8x) than when either molecular recognition method is used in isolation. This integrated "catch-and-latch" approach, which combines the strength and mobility of the fluorous effect with the specificity of base-pairing, provides an additional toolset for DNA nanotechnology, one that enables increased assembly efficiency while requiring significantly fewer DNA sequences. As a result, our integration of fluorous-directed assembly into origami systems represents a cheap, atom-efficient means to produce discrete superstructures.
Topics: Nucleic Acid Conformation; Nanostructures; DNA; Nanotechnology; Base Pairing
PubMed: 36537902
DOI: 10.1021/acsnano.2c10727 -
Nature Communications Nov 2022DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase...
DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase (DddA), and a uracil glycosylase inhibitor to mediate C•G-to-T•A editing in nuclear and organelle DNA. Here we report the development of zinc finger DdCBEs (ZF-DdCBEs) and the improvement of their editing performance through engineering their architectures, defining improved ZF scaffolds, and installing DddA activity-enhancing mutations. We engineer variants with improved DNA specificity by integrating four strategies to reduce off-target editing. We use optimized ZF-DdCBEs to install or correct disease-associated mutations in mitochondria and in the nucleus. Leveraging their small size, we use a single AAV9 to deliver into heart, liver, and skeletal muscle in post-natal mice ZF-DdCBEs that efficiently install disease-associated mutations. While off-target editing of ZF-DdCBEs is likely too high for therapeutic applications, these findings demonstrate a compact, all-protein base editing research tool for precise editing of organelle or nuclear DNA without double-strand DNA breaks.
Topics: Mice; Animals; Gene Editing; CRISPR-Cas Systems; DNA; Zinc Fingers; Cytosine; Mitochondria
PubMed: 36418298
DOI: 10.1038/s41467-022-34784-7 -
Biosensors Jul 2021Surface-enhanced Raman spectroscopy (SERS) merges nanotechnology with conventional Raman spectroscopy to produce an ultrasensitive and highly specific analytical tool... (Review)
Review
Surface-enhanced Raman spectroscopy (SERS) merges nanotechnology with conventional Raman spectroscopy to produce an ultrasensitive and highly specific analytical tool that has been exploited as the optical signal read-out in a variety of advanced applications. In this feature article, we delineate the main features of the intertwined relationship between SERS and nucleic acids (NAs). In particular, we report representative examples of the implementation of SERS in biosensing platforms for NA detection, the integration of DNA as the biorecognition element onto plasmonic materials for SERS analysis of different classes of analytes (from metal ions to microorgniasms) and, finally, the use of structural DNA nanotechnology for the precise engineering of SERS-active nanomaterials.
Topics: Biosensing Techniques; DNA; Gold; Metal Nanoparticles; Nanostructures; Nucleic Acids; Spectrum Analysis, Raman; Surface Properties
PubMed: 34356701
DOI: 10.3390/bios11070230 -
Journal of Translational Medicine Nov 2022Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and...
BACKGROUND
Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.
METHODS
We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes.
RESULTS
We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses.
CONCLUSION
We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.
Topics: Humans; Lentivirus; Retroviridae; Genetic Vectors; Virus Integration; T-Lymphocytes; DNA
PubMed: 36348415
DOI: 10.1186/s12967-022-03729-5