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Chemistry (Weinheim An Der Bergstrasse,... Jun 2024Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of...
Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of ABNO-H-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable 3-fused hexahydropyrrolo-indole conjugates. Similarly modified ABNO-H-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNO-H-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.
PubMed: 38924659
DOI: 10.1002/chem.202402151 -
Current Protocols Jun 2024Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in...
Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.
Topics: In Situ Hybridization, Fluorescence; Humans; Precision Medicine; Paraffin Embedding; Neoplasms; High-Throughput Nucleotide Sequencing; DNA Probes
PubMed: 38923415
DOI: 10.1002/cpz1.1093 -
Physical Chemistry Chemical Physics :... Jun 2024Many fundamental biophysical processes involving gene regulation and gene editing rely, at the molecular level, on an intricate methodology of searching and locating the...
Many fundamental biophysical processes involving gene regulation and gene editing rely, at the molecular level, on an intricate methodology of searching and locating the precise target base pair sequence on the genome by specific binding proteins. A unique mechanism, known as 'facilitated diffusion', which is a combination of 1D sliding along with 3D movement, is considered to be the key step for such events. This also explains the relatively much shorter timescale of the target searching process, compared to other diffusion-controlled biophysical processes. In this work, we aim to probe the modulation of target search dynamics of a protein moiety by estimating the rate of the target search process, and the statistics of the search rounds and timescales accomplished by the 1D and 3D motions, based on first passage time (FPT) calculations. This is studied with its characteristics getting influenced by various given conditions such as, when the DNA is rigid or flexible, and when the target is placed at different locations on the DNA. The current theoretical framework includes a Brownian dynamics simulation setup adopting a straightforward coarse-grained model for a diffusing protein on DNA. Moreover, this theoretical analysis provides insights into the complex target search dynamics by highlighting the significance of the chain dynamics in the mechanistic details of the facilitated diffusion process.
PubMed: 38922594
DOI: 10.1039/d4cp01580k -
Applied Microbiology and Biotechnology Jun 2024The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been...
The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson's r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.
Topics: Humans; Real-Time Polymerase Chain Reaction; Gastrointestinal Microbiome; Feces; Bacteria; Metagenomics; High-Throughput Nucleotide Sequencing; Sensitivity and Specificity; DNA Primers; DNA, Bacterial
PubMed: 38922447
DOI: 10.1007/s00253-024-13204-4 -
Tropical Medicine and Infectious Disease Jun 2024Echinococcosis poses a significant concern in the fields of public health and veterinary care as it can be transmitted between animals and humans. The primary endemic...
Rapid Discriminative Identification of the Two Predominant Echinococcus Species from Canine Fecal Samples in the Tibetan Region of China by Loop-Mediated Isothermal Amplification-Lateral Flow Dipstick Assay.
Echinococcosis poses a significant concern in the fields of public health and veterinary care as it can be transmitted between animals and humans. The primary endemic subtypes are cystic echinococcosis (CE) and alveolar echinococcosis (AE), which result from infestation by and , respectively. A prominent epidemic of echinococcosis greatly affects the Tibet Autonomous Region (TAR) in China. A new technique called the loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) test is introduced in this research to differentiate between and using their repetitive genetic sequences. The test is characterized by its portable nature, simple operation, quick result production, high sensitivity, and low susceptibility to aerosol contamination. The LAMP-LFD method demonstrated an exceptional minimal detection limit, reaching levels as low as approximately 1 fg/μL (femtogram per microliter) of genomic DNA. The assay's specificity was assessed, and no cross-reactivity was seen. A total of 982 dog fecal samples were collected from 54 counties in the TAR region between July 2021 and June 2022. The established method underwent validation using a commercially available ELISA kit. The agreement rate between the LAMP-LFD and ELISA methods was 97.25%, with a sensitivity of 96.05% and a specificity of 97.35%. The assay described in this study improves specificity by using a double-labeled probe, and it reduces the risk of false-positive results caused by aerosol contamination through the use of a sealed device. This makes it a suitable choice for quickly and accurately identifying the two main types of in field settings.
PubMed: 38922048
DOI: 10.3390/tropicalmed9060136 -
Current Issues in Molecular Biology May 2024A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G...
A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.
PubMed: 38920998
DOI: 10.3390/cimb46060326 -
Biosensors May 2024A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living...
A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging.
Topics: Aptamers, Nucleotide; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Biosensing Techniques; Nucleic Acid Hybridization; Fluorescent Dyes
PubMed: 38920578
DOI: 10.3390/bios14060274 -
Nucleic Acids Research Jun 2024Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act...
Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.
PubMed: 38917328
DOI: 10.1093/nar/gkae526 -
Accounts of Chemical Research Jun 2024ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments...
ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments of a DNA cube by Nadrian Seeman in 1991 and a DNA nanomachine by Turberfield and Yurke in 2000 spawned an entire generation of DNA nanodevices ranging from minimalist to rococo architectures. Since our first demonstration in 2009 that a DNA nanodevice can function autonomously inside a living cell, it became clear that this molecular scaffold was well-placed to probe living systems. Its water solubility, biocompatibility, and engineerability to yield molecularly identical assemblies predisposed it to probe and program biology.Since DNA is a modular scaffold, one can integrate independent or interdependent functionalities onto a single assembly. Work from our group has established a new class of organelle-targeted, DNA-based fluorescent reporters. These reporters comprise three to four oligonucleotides that each display a specific motif or module with a specific function. Given the 1:1 stoichiometry of Watson-Crick-Franklin base pairing, all modules are present in a fixed ratio in every DNA nanodevice. These modules include an ion-sensitive dye or a detection module and a normalizing dye for ratiometry that along with detection module forms a "measuring module". The third module is an organelle-targeting module that engages a cognate protein so that the whole assembly is trafficked to the lumen of a target organelle. Together, these modules allow us to measure free ion concentrations with accuracies that were previously unattainable, in subcellular locations that were previously inaccessible, and at single organelle resolution. By revealing that organelles exist in different chemical states, DNA nanodevices are providing new insights into organelle biology. Further, the ability to deliver molecules with cell-type and organelle level precision in animal models is leading to biomedical applications.This Account outlines the development of DNA nanodevices as fluorescent reporters for chemically mapping or modulating organelle function in real time in living systems. We discuss the technical challenges of measuring ions within endomembrane organelles and show how the unique properties of DNA nanodevices enable organelle targeting and chemical mapping. Starting from the pioneering finding that an autonomous DNA nanodevice could map endolysosomal pH in cells, we chart the development of strategies to target organelles beyond the endolysosomal pathway and expanding chemical maps to include all the major ions in physiology, reactive species, enzyme activity, and voltage. We present a series of vignettes highlighting the new biology unlocked with each development, from the discovery of chemical heterogeneity in lysosomes to identifying the first protein importer of Ca into lysosomes. Finally, we discuss the broader applicability of targeting DNA nanodevices organelle-specifically beyond just reporting ions, namely using DNA nanodevices to modulate organelle state, and thereby cell state, with potential therapeutic applications.
PubMed: 38916405
DOI: 10.1021/acs.accounts.4c00191 -
BioRxiv : the Preprint Server For... Jun 2024ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nico-tinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification...
ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nico-tinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hy-drolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for under-standing the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semi-quantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.
PubMed: 38915511
DOI: 10.1101/2024.06.11.598597