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Genomics Nov 2020Effective, sensitive, and reliable diagnostic reagents are of paramount importance for combating the ongoing coronavirus disease 2019 (COVID-19) pandemic when there is...
Effective, sensitive, and reliable diagnostic reagents are of paramount importance for combating the ongoing coronavirus disease 2019 (COVID-19) pandemic when there is neither a preventive vaccine nor a specific drug available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It will cause a large number of false-positive and false-negative tests if currently used diagnostic reagents are undermined. Based on genotyping of 31,421 SARS-CoV-2 genome samples collected up to July 23, 2020, we reveal that essentially all of the current COVID-19 diagnostic targets have undergone mutations. We further show that SARS-CoV-2 has the most mutations on the targets of various nucleocapsid (N) gene primers and probes, which have been widely used around the world to diagnose COVID-19. To understand whether SARS-CoV-2 genes have mutated unevenly, we have computed the mutation rate and mutation h-index of all SARS-CoV-2 genes, indicating that the N gene is one of the most non-conservative genes in the SARS-CoV-2 genome. We show that due to human immune response induced APOBEC mRNA (C > T) editing, diagnostic targets should also be selected to avoid cytidines. Our findings might enable optimally selecting the conservative SARS-CoV-2 genes and proteins for the design and development of COVID-19 diagnostic reagents, prophylactic vaccines, and therapeutic medicines. AVAILABILITY: Interactive real-time online Mutation Tracker.
Topics: COVID-19; COVID-19 Testing; Coronavirus Envelope Proteins; DNA Primers; Genotyping Techniques; Humans; Mutation; Polymorphism, Single Nucleotide; SARS-CoV-2
PubMed: 32966857
DOI: 10.1016/j.ygeno.2020.09.028 -
Micromachines Feb 2021The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019...
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.
PubMed: 33672890
DOI: 10.3390/mi12020197 -
Nature Methods Nov 2022We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by...
We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.
Topics: Animals; Mice; High-Throughput Nucleotide Sequencing; DNA, Complementary; DNA
PubMed: 36216958
DOI: 10.1038/s41592-022-01604-1 -
Angewandte Chemie (International Ed. in... Feb 2022The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among...
The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among various lipids and proteins. Currently, it is still challenging to image these short-lived membrane complexes, especially in living cells. In this work, we present a DNA-based probe, termed "DNA Zipper", which allows the membrane order and pattern of transient interactions to be imaged in living cells using standard fluorescence microscopes. By fine-tuning the length and binding affinity of DNA duplex, these probes can precisely extend the duration of membrane lipid interactions via dynamic DNA hybridization. The correlation between membrane order and the activation of T-cell receptor signaling has also been studied. These programmable DNA probes function after a brief cell incubation, which can be easily adapted to study lipid interactions and membrane order during different membrane signaling events.
Topics: Animals; Cell Membrane; DNA Probes; Dogs; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Madin Darby Canine Kidney Cells
PubMed: 34767659
DOI: 10.1002/anie.202112033 -
Biosensors Feb 2023The prevalence of mutated species of COVID-19 antigens has provided a strong impetus for identifying a cost-effective, rapid and facile strategy for identifying the... (Review)
Review
The prevalence of mutated species of COVID-19 antigens has provided a strong impetus for identifying a cost-effective, rapid and facile strategy for identifying the viral loads in public places. The ever-changing genetic make-up of SARS-CoV-2 posts a significant challenfge for the research community to identify a robust mechanism to target, bind and confirm the presence of a viral load before it spreads. Synthetic DNA constructs are a novel strategy to design complementary DNA sequences specific for antigens of interest as in this review's case SARS-CoV-2 antigens. Small molecules, complementary DNA and protein-DNA complexes have been known to target analytes in minimal concentrations. This phenomenon can be exploited by nanomaterials which have unique electronic properties such as ballistic conduction. Graphene is one such candidate for designing a device with a very low LOD in the order of zeptomolar and attomolar concentrations. Surface modification will be the significant aspect of the device which needs to have a high degree of sensitivity at the same time as providing a rapid signaling mechanism.
Topics: Humans; COVID-19; SARS-CoV-2; Graphite; DNA, Complementary; Biosensing Techniques; Biomarkers
PubMed: 36979519
DOI: 10.3390/bios13030307 -
Frontiers in Cell and Developmental... 2023Cells continuously experience and respond to different physical forces that are used to regulate their physiology and functions. Our ability to measure these mechanical...
Cells continuously experience and respond to different physical forces that are used to regulate their physiology and functions. Our ability to measure these mechanical cues is essential for understanding the bases of various mechanosensing and mechanotransduction processes. While multiple strategies have been developed to study mechanical forces within two-dimensional (2D) cell culture monolayers, the force measurement at cell-cell junctions in real three-dimensional (3D) cell models is still pretty rare. Considering that in real biological systems, cells are exposed to forces from 3D directions, measuring these molecular forces in their native environment is thus highly critical for the better understanding of different development and disease processes. We have recently developed a type of DNA-based molecular probe for measuring intercellular tensile forces in 2D cell models. Herein, we will report the further development and first-time usage of these molecular tension probes to visualize and detect mechanical forces within 3D spheroids and embryoid bodies (EBs). These probes can spontaneously anchor onto live cell membranes via the attached lipid moieties. By varying the concentrations of these DNA probes and their incubation time, we have first characterized the kinetics and efficiency of probe penetration and loading onto tumor spheroids and stem cell EBs of different sizes. After optimization, we have further imaged and measured E-cadherin-mediated forces in these 3D spheroids and EBs for the first time. Our results indicated that these DNA-based molecular tension probes can be used to study the spatiotemporal distributions of target mechanotransduction processes. These powerful imaging tools may be potentially applied to fill the gap between ongoing research of biomechanics in 2D systems and that in real 3D cell complexes.
PubMed: 37920824
DOI: 10.3389/fcell.2023.1220079 -
Molecules (Basel, Switzerland) Oct 2022Conotoxins constitute a treasury of drug resources and have attracted widespread attention. In order to explore biological candidates from the marine cone snail, we...
Conotoxins constitute a treasury of drug resources and have attracted widespread attention. In order to explore biological candidates from the marine cone snail, we isolated and identified three novel conopeptides named as Vi14b, Vi002, Vi003, three conotoxin variants named as Mr3d.1, Mr3e.1, Tx3a.1, and three known conotoxins (Vi15a, Mr3.8 and TCP) from crude venoms of , and Mr3.8 (I-V, II-VI, III-IV) and Tx3a.1 (I-III, II-VI, IV-V) both showed a novel pattern of disulfide connectivity, different from that previously established for the µ- and ψ-conotoxins. Concerning the effect on voltage-gated sodium channels, Mr3e.1, Mr3.8, Tx3a.1, TCP inhibited Na1.4 or Na1.8 by 21.51~24.32% of currents at semi-activated state (TP2) at 10 μmol/L. Certain anti-ovarian cancer effects on ID-8 cells were exhibited by Tx3a.1, Mr3e.1 and Vi14b with IC values of 24.29 µM, 54.97 µM and 111.6 µM, respectively. This work highlights the role of conotoxin libraries in subsequent drug discovery for ovarian cancer treatment.
Topics: Animals; Conotoxins; Conus Snail; DNA, Complementary; Disulfides; Mollusk Venoms; Neoplasms
PubMed: 36235146
DOI: 10.3390/molecules27196609 -
Nature Communications Aug 2019The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there...
The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells. Instead, the signal from affinity-based measurements of these biomolecules depends linearly on the probe concentration. Substituting antibody-based probes tagged for fluorescent quantification with lasing detection probes would create a new platform for biomarker quantification based on optical rather than enzymatic amplification. Here, we construct a virus laser which bridges synthetic biology and laser physics, and demonstrate virus-lasing probes for biosensing. Our virus-lasing probes display an unprecedented > 10,000 times increase in signal from only a 50% increase in probe concentration, using fluorimeter-compatible optics, and can detect biomolecules at sub-100 fmol mL concentrations.
Topics: Antibodies, Monoclonal; Bacteriophage M13; Biophysical Phenomena; Biosensing Techniques; DNA; DNA Probes; Electrons; Fluorescent Dyes; Humans; Lasers; Ligands; Models, Chemical; Nucleic Acids; Oligonucleotide Probes; Polymerase Chain Reaction; Viruses
PubMed: 31399594
DOI: 10.1038/s41467-019-11604-z -
Chembiochem : a European Journal of... Sep 2020DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional...
DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional secret keys in DNA steganography. Brute force testing of different primers will be extremely time consuming, and practically unaffordable when high-throughput sequencing is used. However, the encrypted information can be sequenced and read once the primers are intercepted. A new steganography approach is needed to make the DNA-encoded information safer, if not unhackable. Mixing information-carrying DNA with a partially degenerated DNA library containing single or multiple restriction sites, we have built an additional protective layer that can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA-encrypted information, heating will cause reshuffling and generate endonuclease-resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, DNA steganography possesses both quantum key distribution (QKD)-like function for detecting PCR by an interceptor and a self-destructive property. It is noteworthy that the background noise generated through the protective layer is independent from any sequencing technology including Sanger and high-throughput sequencing. With a DNA ink incorporating the steganography, we have shown that the authenticity of a piece of writing can be confirmed only by authorized persons with knowledge of all embedded keys.
Topics: Computer Security; DNA; DNA Primers; Humans; Polymerase Chain Reaction
PubMed: 32270906
DOI: 10.1002/cbic.202000149 -
Molecules (Basel, Switzerland) Oct 2019Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics... (Review)
Review
Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.
Topics: Aptamers, Nucleotide; DNA Primers; DNA, Single-Stranded; Gene Library; High-Throughput Nucleotide Sequencing; Nucleic Acid Amplification Techniques; Nucleic Acids; Polymerase Chain Reaction; SELEX Aptamer Technique
PubMed: 31591283
DOI: 10.3390/molecules24193598