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Talanta Sep 2022Specific and cost-effective methodologies for human papillomavirus (HPV) gene detection are significant for clinical diagnosis and cancer control. Herein, a label-free...
Specific and cost-effective methodologies for human papillomavirus (HPV) gene detection are significant for clinical diagnosis and cancer control. Herein, a label-free and fluorimetric/colorimetric dual-mode sensing strategy was developed for the quantitative determination of HPV DNA based on the integration of fluorescent DNA-silver nanoclusters (DNA/AgNCs) and G-quadruplex/hemin DNAzyme. The fluorimetric sensing strategy was based on the phenomena that the fluorescence enhancement of DNA/AgNCs obtained in proximity of guanine-rich DNA sequences and the photoinduced electron transfer (PET) effect between the electron donor (DNA/AgNCs) and electron receptor (G-quadruplex/hemin). The colorimetric sensing strategy was relied on the peroxidase-like activity of G-quadruplex/hemin DNAzyme. By integrating DNA/AgNCs and DNAzyme, this dual-mode strategy could produce two independent signals to improve the analytical diversity and accuracy. Under optimized conditions, the fluorimetry and colorimetry of the strategy displayed a linear range of 0.01-4 and 0.02-4 nM, with the low detection limit of 2.3 and 5.2 pM, respectively. Additionally, this dual-mode strategy has been successfully applied to HPV DNA analysis in different real samples with excellent results. Moreover, the sensing platform could be developed for different HPVs DNA assay by only adjusting the recognition sequence, which provided a universal strategy for various kinds of virus analysis.
Topics: Biosensing Techniques; Colorimetry; DNA; DNA, Catalytic; G-Quadruplexes; Hemin; Humans; Nanostructures; Papillomavirus Infections; Silver
PubMed: 35653859
DOI: 10.1016/j.talanta.2022.123554 -
British Journal of Cancer Mar 2020Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for... (Review)
Review
Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for carcinogenesis. Multiple pathways have evolved to protect against both intrinsic and extrinsic genotoxic events, and recent developments have highlighted an unforeseen critical role for RNA in ensuring genome stability. It is currently unclear exactly how RNA molecules participate in the repair pathways, although many models have been proposed and it is possible that RNA acts in diverse ways to facilitate DNA repair. A number of well-documented DNA repair factors have been described to have RNA-binding capacities and, moreover, screens investigating DNA-damage repair mechanisms have identified RNA-binding proteins as a major group of novel factors involved in DNA repair. In this review, we integrate some of these datasets to identify commonalities that might highlight novel and interesting factors for future investigations. This emerging role for RNA opens up a new dimension in the field of DNA repair; we discuss its impact on our current understanding of DNA repair processes and consider how it might influence cancer progression.
Topics: Animals; DNA; DNA Breaks, Double-Stranded; DNA Repair; Humans; RNA
PubMed: 31894141
DOI: 10.1038/s41416-019-0624-1 -
Micron (Oxford, England : 1993) Nov 2022Nanopore-based techniques are widely used owing to their diverse applications such as DNA sequencing, ion detection, gas filtration, protein sequencing, and numerous... (Review)
Review
Nanopore-based techniques are widely used owing to their diverse applications such as DNA sequencing, ion detection, gas filtration, protein sequencing, and numerous other applications. Although commercialized sequencing methods are based on biological nanopores, solid-state nanopore technology is emerging due to its several advantages over biological nanopores, such as its tunable size, chemical and mechanical stability, and possibilities for easy integration with measurement electronics. The unavailability of rapid, low-cost, easy solid-state nanopore fabrication methods with industrial scalability is one of the current bottlenecks in this domain. Among all nanopore fabrication techniques, the Transmission electron microscope (TEM) based fabrication method is frequently used in research labs due to its capability of drilling and tuning nanopores with high accuracy. Given that there are no other methods capable of imaging and fabricating nanopores simultaneously, it is important to discuss the related methods and protocols of TEM. This review focuses on the various aspects of nanopore technology using TEM, from pore fabrication to imaging. Hybrid nanopores are also emerging, which combine the benefits of biological and solid-state nanopores. These can be formed by integrating DNA origami with solid-state nanopores. Creating and imaging DNA origami structures also presents several challenges. We also review DNA origami imaging using conventional TEM. We hope that this review will provide a one-stop reference to TEM applications on solid-state nanopores from fabrication to bioimaging and boost further research in this area.
Topics: DNA; Nanopores; Nanotechnology; Sequence Analysis, DNA
PubMed: 36081256
DOI: 10.1016/j.micron.2022.103347 -
Molecular Cell Nov 2022Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required...
Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA
PubMed: 36272411
DOI: 10.1016/j.molcel.2022.09.030 -
Science (New York, N.Y.) Feb 2021DNA origami is a modular platform for the combination of molecular and colloidal components to create optical, electronic, and biological devices. Integration of such...
DNA origami is a modular platform for the combination of molecular and colloidal components to create optical, electronic, and biological devices. Integration of such nanoscale devices with microfabricated connectors and circuits is challenging: Large numbers of freely diffusing devices must be fixed at desired locations with desired alignment. We present a DNA origami molecule whose energy landscape on lithographic binding sites has a unique maximum. This property enabled device alignment within 3.2° on silica surfaces. Orientation was absolute (all degrees of freedom were specified) and arbitrary (the orientation of every molecule was independently specified). The use of orientation to optimize device performance was shown by aligning fluorescent emission dipoles within microfabricated optical cavities. Large-scale integration was demonstrated with an array of 3456 DNA origami with 12 distinct orientations that indicated the polarization of excitation light.
Topics: Binding Sites; DNA; Nanostructures; Nanotechnology; Nucleic Acid Conformation; Rotation; Silicon Dioxide; Thermodynamics
PubMed: 33602826
DOI: 10.1126/science.abd6179 -
Gene Therapy Jun 2022While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous)...
While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per μg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 10 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.
Topics: Adenoviridae; Animals; DNA; Genetic Vectors; Genomics; Injections, Intravenous; Liver; Mice
PubMed: 34404916
DOI: 10.1038/s41434-021-00278-2 -
Molecules (Basel, Switzerland) Mar 2021The predictable nature of deoxyribonucleic acid (DNA) interactions enables assembly of DNA into almost any arbitrary shape with programmable features of nanometer... (Review)
Review
The predictable nature of deoxyribonucleic acid (DNA) interactions enables assembly of DNA into almost any arbitrary shape with programmable features of nanometer precision. The recent progress of DNA nanotechnology has allowed production of an even wider gamut of possible shapes with high-yield and error-free assembly processes. Most of these structures are, however, limited in size to a nanometer scale. To overcome this limitation, a plethora of studies has been carried out to form larger structures using DNA assemblies as building blocks or tiles. Therefore, DNA tiles have become one of the most widely used building blocks for engineering large, intricate structures with nanometer precision. To create even larger assemblies with highly organized patterns, scientists have developed a variety of structural design principles and assembly methods. This review first summarizes currently available DNA tile toolboxes and the basic principles of lattice formation and hierarchical self-assembly using DNA tiles. Special emphasis is given to the forces involved in the assembly process in liquid-liquid and at solid-liquid interfaces, and how to master them to reach the optimum balance between the involved interactions for successful self-assembly. In addition, we focus on the recent approaches that have shown great potential for the controlled immobilization and positioning of DNA nanostructures on different surfaces. The ability to position DNA objects in a controllable manner on technologically relevant surfaces is one step forward towards the integration of DNA-based materials into nanoelectronic and sensor devices.
Topics: DNA; Nanostructures; Nanotechnology
PubMed: 33801952
DOI: 10.3390/molecules26061502 -
Methods in Molecular Biology (Clifton,... 2022Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development...
Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations. MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.
Topics: Biological Assay; DNA; DNA Methylation; Protein Processing, Post-Translational; Real-Time Polymerase Chain Reaction
PubMed: 35094330
DOI: 10.1007/978-1-0716-1811-0_13 -
Molecular Cell May 2022Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with... (Review)
Review
Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.
Topics: Biophysics; DNA; High-Throughput Nucleotide Sequencing; Molecular Biology; Sequence Analysis, DNA
PubMed: 35561688
DOI: 10.1016/j.molcel.2022.04.024 -
ACS Nano Feb 2020Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels...
Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels of control for artificial systems. As a proof-of-principle, we constructed an RNA-extruding nanofactory using a DNA-origami barrel with an outer diameter of 60 nm as a chassis for integrated rolling-circle transcription and processing of RNA through spatial organization of DNA templates, RNA polymerases, and RNA endonucleases. The incorporation efficiency of molecular components was quantified to be roughly 50% on designed sites within the DNA-origami chassis. Each integrated nanofactory with RNA-producing units, composed of DNA templates and RNA polymerases, produced 100 copies of target RNA in 30 min on average. Further integration of RNA endonucleases that cleave rolling-circle transcripts from concatemers into monomers resulted in 30% processing efficiency. Disabling spatial organization of molecular components on DNA origami resulted in suppression of RNA production as well as processing.
Topics: DNA; DNA-Directed RNA Polymerases; Endoribonucleases; Nanotechnology; Particle Size; RNA; Surface Properties
PubMed: 31922721
DOI: 10.1021/acsnano.9b06466