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Accounts of Chemical Research Oct 2023Nucleophilic addition of bisulfite to pyrimidine bases has been known for a half century, and the reaction has been in use for at least a quarter of a century for...
Nucleophilic addition of bisulfite to pyrimidine bases has been known for a half century, and the reaction has been in use for at least a quarter of a century for identifying 5-methylcytidine in DNA. This account focuses on the chemistry of bisulfite with pseudouridine, an isomer of the RNA nucleoside uridine in which the uracil base is connected to C1' of ribose via C5 instead of N1. Pseudouridine, Ψ, is the most common nucleotide modification found in cellular RNA overall, in part due to its abundance in rRNAs and tRNAs. It has a stabilizing influence on RNA structure because N1 is now available for additional hydrogen bonding and because the heterocycle is slightly better at π stacking. The isomerization of U to Ψ in RNA strands is catalyzed by 13 different enzymes in humans and 11 in ; some of these enzymes are implicated in disease states which is testament to the biological importance of pseudouridine in cells. Recently, pseudouridine came into the limelight as the key modification that, after N1 methylation, enables mRNA vaccines to be delivered efficiently into human tissue with minimal generation of a deleterious immunogenic response. Here we describe the bisulfite reaction with pseudouridine which gives rise to a chemical sequencing method to map the modified base in the epitranscriptome. Unlike the reaction with cytidine, the addition of bisulfite to Ψ leads irreversibly to form an adduct that is bypassed during cDNA synthesis by reverse transcriptases yielding a characteristic deletion signature. Although there were hints to the structure of the bisulfite adduct(s) 30 to 50 years ago, it took modern spectroscopic and computational methods to solve the mystery. Raman spectroscopy along with extensive NMR, ECD, and computational work led to the assignment of the major product as the () diastereomer of an oxygen adduct at C1' of a ring-opened pseudouridine. Mechanistically, this arose from a succession of conjugate addition, E2 elimination, and a [2,3] sigmatropic rearrangement, all of which are stereodefined reactions. A minor reaction with excess bisulfite led to the () isomer of a S-adducted SO group. Understanding structure and mechanism aided the design of a Ψ-specific sequencing reaction and guided attempts to improve the utility and specificity of the method. Separately, we have been investigating the use of nanopore direct RNA sequencing, a single-molecule method that directly analyzes RNA strands isolated from cells after end-ligation of adaptor sequences. By combining the electrical current and base-calling data from the nanopore with dwell-time analysis from the helicase employed to deliver RNA to the nanopore, we were able to map Ψ sites in nearly all sequence contexts. This analysis was employed to find Ψ residues in the SARS-CoV-2 vRNA, to analyze the sequence context effects of mRNA vaccine synthesis via in vitro transcription, and to evaluate the impact of stress on chemical modifications in the ribosome. Most recently, we found that bisulfite treatment of RNA leading to Ψ adducts could modulate the nanopore signal to help in mapping modifications of low occupancy.
Topics: Humans; RNA; Pseudouridine; Nanopore Sequencing; Escherichia coli; COVID-19; SARS-CoV-2; RNA, Ribosomal; RNA Processing, Post-Transcriptional
PubMed: 37700703
DOI: 10.1021/acs.accounts.3c00458 -
Analytical Chemistry Jun 2024Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still...
Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via NA-emplated lick igation hain eaction (DT-CLCR) catalyzed by a eterogeneous anocatalyst made of CuO (hnCuO). This hnCuO-DT-CLCR method is built on two cross-amplifying hnCuO-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCuO-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCuO-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCuO-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCuO-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.
Topics: Copper; Click Chemistry; Nucleic Acid Amplification Techniques; DNA; Catalysis; Humans; Biosensing Techniques; Limit of Detection; DNA, Catalytic; Azides; Colorimetry; Electrochemical Techniques; Cycloaddition Reaction
PubMed: 38853671
DOI: 10.1021/acs.analchem.4c01663 -
Biosensors & Bioelectronics Mar 2024Methylation is one of the most prevalent epigenetic modifications in natural organisms, and the processes of methylation and demethylation are closely associated with...
Methylation is one of the most prevalent epigenetic modifications in natural organisms, and the processes of methylation and demethylation are closely associated with cell growth, differentiation, gene transcription and expression. Abnormal methylation may lead to various human diseases including cancers. Simultaneous analysis of multiple DNA demethylases remains a huge challenge due to the requirement of diverse substrate probes and scarcity of proper signal transduction strategies. Herein, we propose a sensitive and label-free method for simultaneous monitoring of multiple DNA demethylases on the basis of demethylation-activated light-up dual-color RNA aptamers. The presence of targets AlkB homologue-3 (ALKBH3) and fat mass and obesity-associated enzyme (FTO) erases the methyl group in DNA substrate probes, activating the ligation-mediate bidirectional transcription amplification reaction to produce enormous Spinach and Mango aptamers. The resulting RNA aptamers (i.e., Spinach and Mango aptamers) can bind with their cognate nonfluorescent fluorogens (DFHBI and TO1-biotin) to significantly improve the fluorescence signals. This aptamersensor shows high specificity and sensitivity with a limit of detection (LOD) of 8.50 × 10 M for ALKBH3 and 6.80 × 10 M for FTO, and it can apply to screen DNA demethylase inhibitors, evaluate DNA demethylase kinetic parameters, and simultaneously measure multiple endogenous DNA demethylases in a single cell. Importantly, this aptamersensor can accurately discriminate the expressions of ALKBH3 and FTO between healthy tissues and non-small cell lung cancer (NSCLC) patient tissues, offering a powerful platform for clinical diagnosis and drug discovery.
Topics: Humans; RNA; Aptamers, Nucleotide; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Biosensing Techniques; DNA; Demethylation; Lung; AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; Alpha-Ketoglutarate-Dependent Dioxygenase FTO
PubMed: 38147719
DOI: 10.1016/j.bios.2023.115966 -
Nature Structural & Molecular Biology Jun 2024In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair...
In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (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) were recently 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 residue 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 lead to the formation of XLF and X4L4 condensates in vitro, 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: 38898102
DOI: 10.1038/s41594-024-01339-x -
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 -
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 -
Chembiochem : a European Journal of... Mar 2024Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of...
Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2'-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers.
Topics: RNA; Oligonucleotides; DNA; RNA, Small Interfering
PubMed: 38179859
DOI: 10.1002/cbic.202300870 -
Clinical Chemistry and Laboratory... Jan 2024Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early...
OBJECTIVES
Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early cancer detection, however, it is still at its infancy and requires technical improvement. We sought to apply a single-strand DNA sequencing technology, for measuring genetic and fragmentomic features of cfDNA and evaluate the performance in detecting multiple cancers.
METHODS
Blood samples of 364 patients from six cancer types (colorectal, esophageal, gastric, liver, lung, and ovarian cancers) and 675 healthy individuals were included in this study. Circulating tumor DNA mutations, cfDNA fragmentomic features and a set of protein biomarkers were assayed. Sensitivity and specificity were reported by cancer types and stages.
RESULTS
Circular Ligation Amplification and sequencing (CLAmp-seq), a single-strand DNA sequencing technology, yielded a population of ultra-short fragments (<100 bp) than double-strand DNA preparation protocols and reveals a more significant size difference between cancer and healthy cfDNA fragments (25.84 bp vs. 16.05 bp). Analysis of the subnucleosomal peaks in ultra-short cfDNA fragments indicates that these peaks are regulatory element "footprints" and correlates with gene expression and cancer stages. At 98 % specificity, a prediction model using ctDNA mutations alone showed an overall sensitivity of 46 %; sensitivity reaches 60 % when protein is added, sensitivity further increases to 66 % when fragmentomics is also integrated. More improvements observed for samples representing earlier cancer stages than later ones.
CONCLUSIONS
These results suggest synergistic properties of protein, genetic and fragmentomics features in the identification of early-stage cancers.
Topics: Humans; Cell-Free Nucleic Acids; Early Detection of Cancer; Mutation; Circulating Tumor DNA; Neoplasms; Biomarkers, Tumor
PubMed: 37678194
DOI: 10.1515/cclm-2023-0541 -
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