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ECS Sensors Plus Sep 2023In proximity-driven sensing, interactions between a probe and an analyte produce a detectable signal by causing a change in distance of two probe components or signaling...
In proximity-driven sensing, interactions between a probe and an analyte produce a detectable signal by causing a change in distance of two probe components or signaling moieties. By interfacing such systems with DNA-based nanostructures, platforms that are highly sensitive, specific, and programmable can be designed. In this Perspective, we delineate the advantages of using DNA building blocks in proximity-driven nanosensors and provide an overview of recent progress in the field, from sensors that rapidly detect pesticides in food to probes that identify rare cancer cells in blood. We also discuss current challenges and identify key areas that need further development.
PubMed: 37424706
DOI: 10.1149/2754-2726/ace068 -
Nature Communications Aug 2023Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the...
Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.
Topics: Humans; Escherichia coli; High-Throughput Nucleotide Sequencing; RNA-Seq; Anti-Bacterial Agents; DNA Primers; RNA, Ribosomal
PubMed: 37612289
DOI: 10.1038/s41467-023-40137-9 -
Journal of Materials Chemistry. B Aug 2023DNA has excellent molecular recognition properties. At the same time, DNA has a programmable structure, high stability, and can be easily modified, making DNA attractive... (Review)
Review
DNA has excellent molecular recognition properties. At the same time, DNA has a programmable structure, high stability, and can be easily modified, making DNA attractive for biosensor design. To convert DNA hybridization or aptamer binding events to physically detectable signals, various nanomaterials have been extensively exploited to take advantage of their optical and surface properties. A popular sensing scheme is through the adsorption of a fluorescently-labeled DNA probe, where detection is achieved by target-induced probe desorption and fluorescence recovery. Another method is to use DNA to protect the colloidal stability of nanomaterials, where subsequent target binding can decrease the protection ability and induce aggregation; this method has mainly been used for gold nanoparticles. This Perspective summarizes some of our work in examining the sensing mechanisms, and we articulate the importance of the understanding of DNA/surface and target/surface interactions for the development of practical DNA-based biosensors.
Topics: Gold; Metal Nanoparticles; DNA; Biosensing Techniques; Nanostructures
PubMed: 37465903
DOI: 10.1039/d3tb01100c -
Microbiology Spectrum Aug 2023Vaginitis is usually diagnosed empirically, microscopically, via cultures, or by molecular testing for the detection of bacterial vaginosis (BV), vulvovaginal...
Vaginitis is usually diagnosed empirically, microscopically, via cultures, or by molecular testing for the detection of bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), or (TV). The DNA probe-based technique detects BV by identifying , VVC by identifying spp., while real-time PCR-based detection methods identify BV by algorithmic analysis of the absence or presence of known vaginal flora. We examined 8,878 total orders placed for DNA probe-based identification (ID) and 10,464 total orders placed for molecular panel ID. We found that PCR-based BV test positivity reduced from 30% to 23% compared with the population tested with DNA probe-based testing. We also found that PCR-based testing VVC positivity increased from 6.3% and 11.6% when compared with DNA probe-based testing. Bayesian generalized linear analysis estimated a lower mean proportion of positive tests for BV in PCR-based molecular panels than DNA probe testing suggesting an under-call of BV. The same models estimated a higher mean proportion of positive tests for molecular vaginal panels than DNA probe testing suggesting an increased detection of candidal vaginitis. In addition, the mean (SD) age for patients with was 40.5 (40.0-41.1) years. Patients with (now ) were 5.2-8.1 (mean 6.7) years older than patients with . Our retrospective data analysis found that BD Max MVP's ability to discriminate between vaginal candidiasis versus other yeast will help to implement CDC (Centers for Disease Control and Prevention)-recommended treatment options. We also believe that providers' inattention to non-albicans treatment could be an issue nationwide. IMPORTANCE Using retrospective data from U.S. Food and Drug Administration-approved/cleared molecular vaginal panels, molecular methods were found to have higher detection for vaginitis and lower detection for bacterial vaginitis when compared to probe-based methods. In addition, the differentiation of and non- yeast has not reached the physician community as we observed noncompliance in recommended therapy. Furthermore, the pros and cons of migrating to molecular testing from conventional microscopy for identifying bacterial vaginitis and fungal vaginitis have been examined and reported in this paper. Interestingly, the mean (SD) age for patients with was 40.5 (40.0-41.1) years. Patients with were 5.2-8.1 (mean 6.7) years older than patients with .
PubMed: 37615484
DOI: 10.1128/spectrum.01628-23 -
Nature Biotechnology Apr 2024Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed...
Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.
Topics: DNA, Complementary; RNA Isoforms; High-Throughput Nucleotide Sequencing; Protein Isoforms; Sequence Analysis, RNA; Transcriptome; Gene Expression Profiling; RNA
PubMed: 37291427
DOI: 10.1038/s41587-023-01815-7 -
Chemistry (Weinheim An Der Bergstrasse,... Aug 2023Accurate cancer diagnosis especially early diagnosis is of great importance for prompt therapy and elevated survival rate. mRNAs are widely used as biomarkers for cancer...
Accurate cancer diagnosis especially early diagnosis is of great importance for prompt therapy and elevated survival rate. mRNAs are widely used as biomarkers for cancer identification and treatment. mRNA expression levels are highly associated with cancer stage and malignant progression. Nevertheless, single type mRNA detection is insufficient and unreliable. Herein, we developed a DNA nano-windmill probe for in situ multiplexed mRNAs detection and imaging in this paper. The probe is designed to simultaneously target four types of mRNA through wind blades. Importantly, recognition of targets is independent from each other, which further facilitate cell type discrimination. The probe can specifically distinguish cancer cell lines from normal cells. In addition, it can identify changes in mRNA expression levels in living cells. The current strategy enriches the toolbox for improving the accuracy of cancer diagnosis and therapeutic solutions.
Topics: RNA, Messenger; DNA Probes; Cell Line, Tumor; DNA
PubMed: 37314386
DOI: 10.1002/chem.202301300 -
Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507 -
Nature Communications Nov 2023Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex...
Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.
Topics: DNA, Viral; Integrases; Retroviridae; Spumavirus; DNA, Complementary; Virus Integration
PubMed: 37923737
DOI: 10.1038/s41467-023-42641-4 -
Plants (Basel, Switzerland) Jul 2023Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem... (Review)
Review
Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem repeats, and distributed repetitive sequence probe-based FISH, have certain limitations, including difficulties in probe synthesis, low sensitivity, cross-hybridization, and limited resolution. In contrast, oligo-based FISH represents a more efficient method for chromosomal studies in plants. Oligo probes are computationally designed and synthesized for any plant species with a sequenced genome and are suitable for single and repetitive DNA sequences, entire chromosomes, or chromosomal segments. Furthermore, oligo probes used in the FISH experiment provide high specificity, resolution, and multiplexing. Moreover, oligo probes made from one species are applicable for studying other genetically and taxonomically related species whose genome has not been sequenced yet, facilitating molecular cytogenetic studies of non-model plants. However, there are some limitations of oligo probes that should be considered, such as requiring prior knowledge of the probe design process and FISH signal issues with shorter probes of background noises during oligo-FISH experiments. This review comprehensively discusses de novo oligo probe synthesis with more focus on single-copy DNA sequences, preparation, improvement, and factors that affect oligo-FISH efficiency. Furthermore, this review highlights recent applications of oligo-FISH in a wide range of plant chromosomal studies.
PubMed: 37570972
DOI: 10.3390/plants12152816 -
BioRxiv : the Preprint Server For... Dec 2023Mechanical forces play an important role in cellular communication and signaling. We developed in this study novel electrochemical DNA-based force sensors for measuring...
Mechanical forces play an important role in cellular communication and signaling. We developed in this study novel electrochemical DNA-based force sensors for measuring cell-generated adhesion forces. Two types of DNA probes, i.e., tension gauge tether and DNA hairpin, were constructed on the surface of a smartphone-based electrochemical device to detect piconewton-scale cellular forces at tunable levels. Upon experiencing cellular tension, the unfolding of DNA probes induces the separation of redox reporters from the surface of the electrode, which results in detectable electrochemical signals. Using integrin-mediated cell adhesion as an example, our results indicated that these electrochemical sensors can be used for highly sensitive, robust, simple, and portable measurement of cell-generated forces.
PubMed: 38106148
DOI: 10.1101/2023.12.03.569814