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ACS Synthetic Biology Sep 2023Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but...
Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.
Topics: High-Throughput Nucleotide Sequencing; DNA
PubMed: 37582217
DOI: 10.1021/acssynbio.3c00183 -
International Journal of Biological... Aug 2023RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a...
RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 μM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.
Topics: Humans; Transcription Factors; Cell Line; Neoplasms; Epigenesis, Genetic; Repressor Proteins
PubMed: 37399862
DOI: 10.1016/j.ijbiomac.2023.125632 -
Biochemical and Biophysical Research... Feb 2024DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close...
DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a "synapsis-like" DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.
Topics: Humans; DNA; DNA Repair; DNA-Binding Proteins; Endodeoxyribonucleases; Exodeoxyribonucleases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38217957
DOI: 10.1016/j.bbrc.2023.149464 -
Scientific Reports Sep 2023DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this...
DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this work, we propose composite motifs, a framework that uses a mixture of prefabricated motifs as building blocks to reduce synthesis cost by scaling logical density. To write data, we introduce Bridge Oligonucleotide Assembly, an enzymatic ligation technique for synthesizing oligos based on composite motifs. To sequence data, we introduce Direct Oligonucleotide Sequencing, a nanopore-based technique to sequence short oligos, eliminating common preparatory steps like DNA assembly, amplification and end-prep. To decode data, we introduce Motif-Search, a novel consensus caller that provides accurate reconstruction despite synthesis and sequencing errors. Using the proposed methods, we present an end-to-end experiment where we store the text "HelloWorld" at a logical density of 84 bits/cycle (14-42× improvement over state-of-the-art).
Topics: DNA; Oligonucleotides; Consensus; Nanopores; Nutritional Status
PubMed: 37749195
DOI: 10.1038/s41598-023-43172-0 -
Journal of Biological Methods 2023Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a...
Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.
PubMed: 38023773
DOI: 10.14440/jbm.2023.411 -
Genes To Cells : Devoted To Molecular &... Aug 2023The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination...
The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double-strand (ds) DNA-dependent 3'-5' exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt-ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3' protruding ends; however, the addition of single-strand DNA-targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.
Topics: Cloning, Molecular; DNA; Escherichia coli; Homologous Recombination; DNA, Single-Stranded; Plasmids
PubMed: 37132531
DOI: 10.1111/gtc.13034 -
BioRxiv : the Preprint Server For... Jul 2023In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in...
In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.
PubMed: 37503201
DOI: 10.1101/2023.07.12.548668 -
Viruses Jul 2023Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular...
Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis-hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by I digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors.
Topics: Animals; Chickens; Serogroup; Communicable Diseases; Hepatitis A; Adenoviridae; Capsid Proteins; DNA, Viral
PubMed: 37632000
DOI: 10.3390/v15081657 -
GDF15 restrains myocardial ischemia-reperfusion injury through inhibiting GPX4 mediated ferroptosis.Aging Jan 2024Growth and differentiation factor 15 (GDF15) has been proved to regulate the process of Myocardial ischemia-reperfusion injury (MIRI), which is a serious complication of...
BACKGROUND
Growth and differentiation factor 15 (GDF15) has been proved to regulate the process of Myocardial ischemia-reperfusion injury (MIRI), which is a serious complication of reperfusion therapy. The present study aimed to explore if GDF15 could regulate the MIRI-induced ferroptosis.
METHOD
MIRI animal model was established by ligating the left anterior descending coronary artery. Oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to imitate MIRI . The indicators of ferroptosis including mitochondrial damage, GPX4, FACL4, XCT4, and oxidative stress markers were evaluated.
RESULTS
Overexpression of GDF15 greatly inhibited MIRI, improved cardiac function, alleviated MIRI-induced ferroptosis. pc-DNA-GDF15 significantly inhibited the oxidative stress condition and inflammation response. The OGD/R-induced ferroptosis was also inhibited by pc-DNA-GDF15.
CONCLUSION
We proved that the MIRI-induced ferroptosis could by inhibited by pc-DNA-GDF15 through evaluating mitochondrial damage, MDA, GSH, and GSSG. Our research provides a new insight for the prevention and treatment of MIRI, and a new understanding for the mechanism of MIRI-induced ferroptosis.
Topics: Animals; Coronary Vessels; DNA; Ferroptosis; Glucose; Growth Differentiation Factor 15; Myocardial Reperfusion Injury; Oxygen
PubMed: 38206295
DOI: 10.18632/aging.205402 -
Cancers Dec 2023Patients diagnosed with epithelial ovarian cancer may undergo reflex tumour / testing followed by germline testing in patients with a positive tumour test result. This...
Patients diagnosed with epithelial ovarian cancer may undergo reflex tumour / testing followed by germline testing in patients with a positive tumour test result. This testing model relies on tumour / tests being able to detect all types of pathogenic variant. We analysed germline and tumour test results from patients treated for epithelial ovarian cancer at our specialist oncological referral centre. Tumour testing was performed using the next-generation sequencing (NGS)-based myChoice companion diagnostic (CDx; Myriad Genetics, Inc.). Germline testing was performed in the North West Genomic Laboratory Hub using NGS and multiplex ligation-dependent probe amplification. Between 11 April 2021 and 11 October 2023, 382 patients were successfully tested for tumour and variants. Of these, 367 (96.1%) patients were tested for germline / variants. In those patients who underwent tumour and germline testing, 15.3% (56/367) had a / pathogenic variant (36 germline and 20 somatic). All germline pathogenic small sequencing variants were detected in tumour DNA. By contrast, 3 out of 8 germline pathogenic large rearrangements were not reported in tumour DNA. The overall concordance of germline pathogenic variants detected in germline and tumour DNA was clinically acceptable at 91.7% (33/36). The myChoice CDx was able to detect most germline pathogenic variants in tumour DNA, although a proportion of pathogenic large rearrangements were not reported. If Myriad's myChoice CDx is used for tumour testing, our data supports a testing strategy of germline and tumour testing in all patients diagnosed with epithelial ovarian cancer aged < 79 years old, with germline testing only necessary for patients aged ≥ 80 years old with a tumour pathogenic variant.
PubMed: 38201604
DOI: 10.3390/cancers16010177