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Advanced Science (Weinheim,... Nov 2023DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot...
DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot be actively processed in DNA pools. An active DNA data editing process is developed using splint ligation in a droplet-controlled fluidics (DCF) system. DNA fragments of discrete sizes (100-500 bps) are synthesized for droplet assembly, and programmed sequence information exchange occurred. The encoded DNA sequences are processed in series and parallel to synthesize the determined DNA pools, enabling random access using polymerase chain reaction amplification. The sequencing results of the assembled DNA data pools can be orderly aligned for decoding and have high fidelity through address primer scanning. Furthermore, eight 90 bps DNA pools with pixel information (png: 0.27-0.28 kB), encoded by codons, are synthesized to create eight 270 bps DNA pools with an animation movie chip file (mp4: 12 kB) in the DCF system.
Topics: DNA; Polymerase Chain Reaction
PubMed: 37755129
DOI: 10.1002/advs.202303197 -
Cell Death Discovery Oct 2023Maladaptive repair of acute kidney injury (AKI) is associated with a high risk of developing chronic kidney disease deemed irremediable even in present days. When AKI...
Maladaptive repair of acute kidney injury (AKI) is associated with a high risk of developing chronic kidney disease deemed irremediable even in present days. When AKI arises from ischemia-reperfusion injury, hypoxia usually plays a major role. Although both hypoxia-inducible factor-1α (HIF-1α) and yes-associated protein (YAP) have been proven to promote renal cell survival under hypoxia, there is a lack of research that studies the crosstalk of the two and its effect on kidney repair. In studying the crosstalk, CoCl was used to create a mimetic hypoxic environment. Immunoprecipitation and proximity ligation assays were performed to verify protein interactions. The results show that HIF-1α interacts with YAP and promotes nuclear translocation of YAP at a high cell density under hypoxic conditions, suggesting HIF-1α serves as a direct carrier that enables YAP nuclear translocation. This is the first study to identify HIF-1α as a crucial pathway for YAP nuclear translocation under hypoxic conditions. Once translocated into a nucleus, YAP protects cells from DNA damage and apoptosis under hypoxic conditions. Since it is unlikely for YAP to translocate into a nucleus without HIF-1α, any treatment that fosters the crosstalk between the two holds the potential to improve cell recovery from hypoxic insults.
PubMed: 37863897
DOI: 10.1038/s41420-023-01687-5 -
Methods (San Diego, Calif.) Oct 2023While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic...
While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic degradation, potential immunogenicity, and delivery issues, which can limit their applications. Peptide Nucleic Acids (PNAs) are promising alternatives due to their high affinity for DNA and RNA, the high resistance to enzymatic degradation, and the easy introduction of a wide range of potential modifications. Chemical modifications that enable the covalent targeting of specific DNA and RNA strands offer additional advantages, including enhanced potency. The current study focuses on the utilization of furan-PNAs as pro-reactive probe systems and their applications to DNA and RNA targeting. Specifically, in this methodological paper, we provide practical insights into the design, synthesis, and application of furan-containing PNA probes for achieving efficient PNA-DNA and PNA-RNA interstrand crosslinking (ICL), as well as ON-templated PNA-PNA ligation systems. Furthermore, we discuss the applications of these probes in targeting DNA secondary structures, such as G-quadruplexes and i-motifs, target pull-down assays, and on-surface detection.
Topics: Nucleic Acids; Peptide Nucleic Acids; RNA; Oligonucleotides; Furans
PubMed: 37604247
DOI: 10.1016/j.ymeth.2023.08.010 -
Scientific Reports May 2024Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of...
Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.
Topics: SARS-CoV-2; Software; Polymerase Chain Reaction; Oligonucleotides; COVID-19; Computational Biology; DNA, Single-Stranded
PubMed: 38750104
DOI: 10.1038/s41598-024-59497-3 -
BioRxiv : the Preprint Server For... Feb 2024Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication...
Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.
PubMed: 37662334
DOI: 10.1101/2023.08.22.554340 -
Theranostics 2023Increasing evidence suggests that hemodynamic disturbed flow induces endothelial dysfunction via a complex biological process so-called endothelial to mesenchymal...
Increasing evidence suggests that hemodynamic disturbed flow induces endothelial dysfunction via a complex biological process so-called endothelial to mesenchymal transition (EndoMT). Recently, DNA methyltransferases (DNMTs) was reported as a key molecular mediator to promote EndoMT. Our understanding of how DNMTs, particularly the maintenance DNMTs, DNMT1, coordinate EndoMT is still lacking. A parallel-plate flow apparatus and perfusion devices were used to apply fluid with endothelial protective pulsatile shear (PS, to mimic the laminar flow) or harmful oscillatory shear (OS, to mimic the disturbed flow) to cultured endothelial cells (ECs). Endothelial lineage tracing mice and conditional EC Dnmt1 knockout mice were subjected to a surgery of carotid partial ligation to generate the flow-accelerated atherogenesis models. Western blotting, quantitative RT-PCR, immunofluorescent staining, methylation-specific PCR, chromatin immunoprecipitation, endothelial functional assays, and assessments for neointimal formation and atherosclerosis were performed. Inhibition of DNMTs with 5-aza-2'-deoxycytidine (5-Aza) suppressed the disturbed flow/OS-induced EndoMT, both in cultured cells and the endothelial lineage tracing mice. 5-Aza also ameliorated the downregulation of aldehyde dehydrogenases (ALDHs) and β-alanine biosynthesis caused by disturbed flow/OS. Knockdown of the ALDH family proteins, ALDH2, ALDH3A1, and ALDH6A1, showed an EndoMT-induction effect as OS. Supplementation of cells with the functional metabolites of β-alanine, carnosine and acetyl-CoA (acetate), reversed EndoMT, likely via inhibiting the phosphorylation of Smad2/3. Endothelial-specific knockout of Dnmt1 protected the vasculature from disturbed flow-induced remodeling and atherosclerosis. Endothelial DNMT1 acts as one of the key epigenetic factors to mediate the hemodynamically regulated EndoMT at least through repressing the expression of ALDH2, ALDH3A1, and ALDH6A1. Supplementation with carnosine and acetate may have a great potential in the prevention and treatment of atherosclerosis.
Topics: Animals; Mice; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Atherosclerosis; Azacitidine; Carnosine; DNA Modification Methylases; Endothelial Cells; Homeostasis; DNA (Cytosine-5-)-Methyltransferase 1
PubMed: 37649604
DOI: 10.7150/thno.84427 -
Proceedings of the National Academy of... Aug 2023Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an FeS cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.
Topics: Humans; SARS-CoV-2; COVID-19 Drug Treatment; COVID-19; DNA Helicases; RNA; Sulfur; Viral Nonstructural Proteins; RNA Helicases
PubMed: 37552760
DOI: 10.1073/pnas.2303860120 -
ACS Synthetic Biology Oct 2023Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or...
Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or double-stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product recircularization using restriction and ligation, leading to a higher amplification yield and enabling the generation of circular products. However, C2CA is a multistep, nonisothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37 °C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.
Topics: DNA; DNA, Circular; Nucleic Acid Amplification Techniques
PubMed: 37729629
DOI: 10.1021/acssynbio.3c00390 -
Frontiers in Bioengineering and... 2023Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory...
Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.
PubMed: 37635997
DOI: 10.3389/fbioe.2023.1167534 -
Journal of Molecular Histology Feb 2024Sepsis has a systemic inflammatory response syndrome caused by infection. While neutrophils play contradictory roles in different stages of sepsis. Neutrophils have been...
Sepsis has a systemic inflammatory response syndrome caused by infection. While neutrophils play contradictory roles in different stages of sepsis. Neutrophils have been proven to play an antibacterial role by producing neutrophil extracellular traps (NETs). Although the NET is beneficial to bacteria resistance, abnormal NET increases tissue damage. The complement C5a receptor 1 (C5ar1) is a gene related to strong inflammatory reactions and is found to be associated with inflammatory factors. This study found that there were 45 down-regulated genes and 704 up-regulated genes in sepsis rats by transcriptome sequencing. And those genes were significantly related to inflammation and immunity by GO and KEGG enrichment analysis involving the chemokine signaling pathway, the Toll-like receptor (TLR) signaling pathway, and the Fc gamma R-mediated phagocytosis. Additionally, the C5ar1 gene was significantly upregulated with interesting potential in sepsis and used for further study. This study used cecum ligation and puncture (CLP) rats that were respectively injected intravenously with PBS or the lentivirus vector to explore the effect of C5ar1 on CLP rats. It demonstrated that silenced- C5ar1 inhibited the ALT, AST, BUN, and CREA levels, improved the lung and spleen injury, and reduced the TNF-α, IL-6, IL-1β, IL-10, cf-DNA, and cfDNA/MPO levels. Additionally, silenced C5ar1 inhibited the TLR2, TLR4, and peptidylarginine deiminase 4 expression levels, which suggested the improvement of silenced C5ar1 on sepsis via inhibiting NETs and the TLR signaling pathway. This study provides a basis and new direction for the study of treatment on sepsis.
Topics: Rats; Animals; Extracellular Traps; Neutrophils; Inflammation; Lung; Sepsis
PubMed: 38165570
DOI: 10.1007/s10735-023-10172-3