-
Electrophoresis May 2023A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for...
A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2 µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.
Topics: Humans; Microfluidics; Lab-On-A-Chip Devices; Polymerase Chain Reaction; DNA; Solid Phase Extraction; Microfluidic Analytical Techniques
PubMed: 36694428
DOI: 10.1002/elps.202200185 -
Chemical Society Reviews May 2016The combination of nanostructures with biomolecules leading to the generation of functional nanosystems holds great promise for biotechnological and biomedical... (Review)
Review
The combination of nanostructures with biomolecules leading to the generation of functional nanosystems holds great promise for biotechnological and biomedical applications. As a naturally occurring biomacromolecule, DNA exhibits excellent biocompatibility and programmability. Also, scalable synthesis can be readily realized through automated instruments. Such unique properties, together with Watson-Crick base-pairing interactions, make DNA a particularly promising candidate to be used as a building block material for a wide variety of nanostructures. In the past few decades, various DNA nanostructures have been developed, including one-, two- and three-dimensional nanomaterials. Aptamers are single-stranded DNA or RNA molecules selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), with specific recognition abilities to their targets. Therefore, integrating aptamers into DNA nanostructures results in powerful tools for biosensing and bioimaging applications. Furthermore, owing to their high loading capability, aptamer-modified DNA nanostructures have also been altered to play the role of drug nanocarriers for in vivo applications and targeted cancer therapy. In this review, we summarize recent progress in the design of aptamers and related DNA molecule-integrated DNA nanostructures as well as their applications in biosensing, bioimaging and cancer therapy. To begin with, we first introduce the SELEX technology. Subsequently, the methodologies for the preparation of aptamer-integrated DNA nanostructures are presented. Then, we highlight their applications in biosensing and bioimaging for various targets, as well as targeted cancer therapy applications. Finally, we discuss several challenges and further opportunities in this emerging field.
Topics: Animals; Aptamers, Nucleotide; Biosensing Techniques; DNA; Humans; Molecular Imaging; Nanotechnology; Neoplasms
PubMed: 26954935
DOI: 10.1039/c5cs00645g -
Nature Aug 2022Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a...
Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.
Topics: CRISPR-Cas Systems; DNA; Gene Editing; Gene Expression; Genome; Information Storage and Retrieval; Integrases; Prokaryotic Cells; RNA; Reverse Transcription; Time Factors
PubMed: 35896746
DOI: 10.1038/s41586-022-04994-6 -
The Plant Journal : For Cell and... Jan 2022Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes...
Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.
Topics: Agrobacterium tumefaciens; Arabidopsis; DNA, Bacterial; DNA, Plant; DNA-Directed DNA Polymerase; Gene Targeting; Homologous Recombination; Plants, Genetically Modified; Transgenes
PubMed: 34713516
DOI: 10.1111/tpj.15557 -
ACS Nano Jan 2023An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges...
An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges of DNA origami tiles we control their hierarchical assembly via fluorous-directed recognition. When we combine this approach with Watson-Crick base-pairing we form discrete dimeric constructs in significantly higher yield (8x) than when either molecular recognition method is used in isolation. This integrated "catch-and-latch" approach, which combines the strength and mobility of the fluorous effect with the specificity of base-pairing, provides an additional toolset for DNA nanotechnology, one that enables increased assembly efficiency while requiring significantly fewer DNA sequences. As a result, our integration of fluorous-directed assembly into origami systems represents a cheap, atom-efficient means to produce discrete superstructures.
Topics: Nucleic Acid Conformation; Nanostructures; DNA; Nanotechnology; Base Pairing
PubMed: 36537902
DOI: 10.1021/acsnano.2c10727 -
Sensors (Basel, Switzerland) Nov 2020Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard... (Review)
Review
Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard pesticide analysis based on mass spectrometry takes a lot of time, money and effort. Thus, simple, reliable, cost-effective and field applicable methods for pesticide detection have been actively developed. One of the most promising technologies is an aptamer-based biosensor or so-called aptasensor. It utilizes aptamers, short single-stranded DNAs or RNAs, as pesticide recognition elements to integrate with various innovative biosensing technologies for specific and sensitive detection of pesticide residues. Several platforms for aptasensors have been dynamically established, such as colorimetry, fluorometry, electrochemistry, electrochemiluminescence (ECL) and so forth. Each platform has both advantages and disadvantages depending on the purpose of use and readiness of technology. For example, colorimetric-based aptasensors are more affordable than others because of the simplicity of fabrication and resource requirements. Electrochemical-based aptasensors have mainly shown better sensitivity than others with exceedingly low detection limits. This paper critically reviews the progression of pesticide aptasensors throughout the development process, including the selection, characterization and modification of aptamers, the conceptual frameworks of integrating aptamers and biosensors, the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users) criteria of different platforms and the future outlook.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Colorimetry; DNA, Single-Stranded; Pesticides
PubMed: 33260648
DOI: 10.3390/s20236809 -
Progress in Biophysics and Molecular... Oct 2019In vertebrates, double-strand breaks in DNA are primarily repaired by Non-Homologous End-Joining (NHEJ). The ring-shaped Ku heterodimer rapidly senses and threads onto... (Review)
Review
In vertebrates, double-strand breaks in DNA are primarily repaired by Non-Homologous End-Joining (NHEJ). The ring-shaped Ku heterodimer rapidly senses and threads onto broken DNA ends forming a recruiting hub. Through protein-protein contacts eventually reinforced by protein-DNA interactions, the Ku-DNA hub attracts a series of specialized proteins with scaffolding and/or enzymatic properties. To shed light on these dynamic interplays, we review here current knowledge on proteins directly interacting with Ku and on the contact points involved, with a particular accent on the different classes of Ku-binding motifs identified in several Ku partners. An integrated structural model of the core NHEJ network at the synapsis step is proposed.
Topics: Amino Acid Motifs; Animals; DNA; DNA End-Joining Repair; Humans; Ku Autoantigen
PubMed: 30851288
DOI: 10.1016/j.pbiomolbio.2019.03.001 -
Current Issues in Molecular Biology 2020Modern genomic sequencing and bioinformatics approaches have detected numerous examples of DNA sequences derived from DNA and RNA virus genomes integrated into both... (Review)
Review
Modern genomic sequencing and bioinformatics approaches have detected numerous examples of DNA sequences derived from DNA and RNA virus genomes integrated into both vertebrate and insect genomes. Retroviruses encode RNA-dependent DNA polymerases (reverse transcriptases) and integrases that convert their RNA viral genomes into DNA proviruses and facilitate proviral DNA integration into the host genome. Surprisingly, DNA sequences derived from RNA viruses that do not encode these enzymes also occur in host genomes. Non-retroviral integrated RNA virus sequences (NIRVS) occur at relatively high frequency in the genomes of the arboviral vectors and , are not distributed randomly and possibly contribute to mosquito antiviral immunity, suggesting these mosquitoes could serve as a model system for unravelling the function of NIRVS. Here we address the following questions: What drives DNA synthesis from the genomes of non-retroviral RNA viruses? How does integration of virus cDNA into host DNA occur, and what is its biological function (if any)? We review current knowledge of viral integrations in insect genomes, hypothesize mechanisms of NIRVS formation and their potential impact on insect biology, particularly antiviral immunity, and suggest directions for future research.
Topics: Aedes; Animals; Computational Biology; DNA Viruses; Endogenous Retroviruses; Genome, Insect; Genomics; Host-Pathogen Interactions; Insecta; Mosquito Vectors; RNA Viruses; RNA, Small Interfering; Retroelements; Virus Integration
PubMed: 31167954
DOI: 10.21775/cimb.034.013 -
Acta Biochimica Et Biophysica Sinica May 2022The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During...
The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety.
Topics: CRISPR-Cas Systems; DNA; DNA Breaks, Double-Stranded; DNA Repair; Gene Editing
PubMed: 35643959
DOI: 10.3724/abbs.2022056 -
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