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Methods in Molecular Biology (Clifton,... 2022PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any...
PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.
Topics: Cloning, Molecular; DNA; Gene Library; Mutagenesis; Plasmids; Polymerase Chain Reaction
PubMed: 35727447
DOI: 10.1007/978-1-0716-2152-3_8 -
International Journal of Molecular... Aug 2022Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of...
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.
Topics: CRISPR-Cas Systems; DNA; Gene Editing; Gene Rearrangement; Plants; Plasmids
PubMed: 35955778
DOI: 10.3390/ijms23158636 -
Nanoscale Horizons May 2023Integrating rationally designed DNA molecular walkers and DNA origami platforms is a promising route towards advanced nano-robotics of diverse functions. Unleashing the...
Integrating rationally designed DNA molecular walkers and DNA origami platforms is a promising route towards advanced nano-robotics of diverse functions. Unleashing the full potential in this direction requires DNA walker-origami systems beyond the present simplistic bridge-burning designs for automated repeatable operation and scalable nano-robotic functions. Here we report such a DNA walker-origami system integrating an advanced light-powered DNA bipedal walker and a ∼170 nm-long rod-like DNA origami platform. This light-powered walker is fully qualified as a genuine translational molecular motor, and relies entirely on pure mechanical effects that are complicated by the origami surface but must be preserved for the walker's proper operation. This is made possible by tailor-designing the origami for optimal match with the walker to best preserve its core mechanics. A new fluorescence method is combined with site-controlled motility experiments to yield distinct and reliable signals for the walker's self-directed and processive motion despite origami-complicated fluorophore emission. The resultant integrated DNA walker-origami system provides a 'seed' system for future development of advanced light-powered DNA nano-robots (, for scalable walker-automated chemical synthesis), and also truly bio-mimicking nano-muscles powered by genuine artificial translational molecular motors.
Topics: Nanotechnology; DNA; Motion; Fluorescent Dyes; Robotics
PubMed: 37038716
DOI: 10.1039/d2nh00565d -
BioTechniques May 2023Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and...
Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for and delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.
Topics: CRISPR-Cas Systems; Workflow; Gene Editing; Genetic Therapy; DNA
PubMed: 37161298
DOI: 10.2144/btn-2023-0013 -
Nature Materials Sep 2021Recently, DNA has been used to make nanodevices for a myriad of applications across fields including medicine, nanomanufacturing, synthetic biology, biosensing and...
Recently, DNA has been used to make nanodevices for a myriad of applications across fields including medicine, nanomanufacturing, synthetic biology, biosensing and biophysics. However, current DNA nanodevices rely primarily on geometric design, and it remains challenging to rationally design functional properties such as force-response or actuation behaviour. Here we report an iterative design pipeline for DNA assemblies that integrates computer-aided engineering based on coarse-grained molecular dynamics with a versatile computer-aided design approach that combines top-down automation with bottom-up control over geometry. This intuitive framework allows for rapid construction of large, multicomponent assemblies from three-dimensional models with finer control over the geometrical, mechanical and dynamical properties of the DNA structures in an automated manner. This approach expands the scope of structural complexity and enhances mechanical and dynamic design of DNA assemblies.
Topics: Computer-Aided Design; DNA; Microscopy, Electron, Transmission; Molecular Dynamics Simulation; Nanostructures; Nanotechnology
PubMed: 33875848
DOI: 10.1038/s41563-021-00978-5 -
International Journal of Molecular... Jan 2020The transfer of genetic material from the mitochondria and plastid to the nucleus gives rise to nuclear integrants of mitochondrial DNA (NUMTs) and nuclear integrants of... (Review)
Review
The transfer of genetic material from the mitochondria and plastid to the nucleus gives rise to nuclear integrants of mitochondrial DNA (NUMTs) and nuclear integrants of plastid DNA (NUPTs). This frequently occurring DNA transfer is ongoing and has important evolutionary implications. In this review, based on previous studies and the analysis of NUMT/NUPT insertions of more than 200 sequenced plant genomes, we analyzed and summarized the general features of NUMTs/NUPTs and highlighted the genetic consequence of organellar DNA insertions. The statistics of organellar DNA integrants among various plant genomes revealed that organellar DNA-derived sequence content is positively correlated with the nuclear genome size. After integration, the nuclear organellar DNA could undergo different fates, including elimination, mutation, rearrangement, fragmentation, and proliferation. The integrated organellar DNAs play important roles in increasing genetic diversity, promoting gene and genome evolution, and are involved in sex chromosome evolution in dioecious plants. The integrating mechanisms, involving non-homologous end joining at double-strand breaks were also discussed.
Topics: Cell Nucleus; Cell Proliferation; DNA End-Joining Repair; DNA, Chloroplast; DNA, Mitochondrial; Evolution, Molecular; Genome Size; Genome, Plant; Mitochondria; Mutation; Plants; Plastids; Sex Chromosomes
PubMed: 31973163
DOI: 10.3390/ijms21030707 -
Accounts of Chemical Research Sep 2019Nanoparticles (NPs) have enormous potential to improve disease diagnosis and treatment due to their intrinsic electronic, optical, magnetic, mechanical, and... (Review)
Review
Nanoparticles (NPs) have enormous potential to improve disease diagnosis and treatment due to their intrinsic electronic, optical, magnetic, mechanical, and physiological properties. To realize their full potential for nanomedicine, NPs must be biocompatible and targetable toward specific biomolecules to ensure selective sensing, imaging, and drug delivery in complex environments such as living cells, tissues, animals, and human bodies. In this Account, we summarize our efforts to impart specific biocompatibility and biorecognition functionality to NPs by developing strategies to integrate inorganic and organic NPs with functional DNA (fDNA), including aptamers, DNAzymes, and aptazymes to create fDNA-NPs. These hybrid NPs take advantage of fDNA's ability to either bind targets or catalyze reactions in the presence of targets selectively and utilize their unique physicochemical properties including small size, low immunogenicity, and ease of synthesis and chemical modification in comparison with other molecules such as antibodies. By integrating inorganic NPs such as gold NPs, quantum dots, and iron oxide nanoparticles with fDNA, we designed stimuli-responsive fDNA-NPs that exhibit target induced assembly and disassembly of NPs, resulting in a variety of colorimetric, fluorescent, and magnetic resonance imaging (MRI)-based sensors for diagnostic of a broad range of analytes. To impart both biocompatibility and selectivity on inorganic NPs for targeted bioimaging, we have demonstrated DNA-mediated surface functionalization, shape-controlled synthesis, and coordinative assembly of such NPs as specific bioprobes. A highlight is provided on the construction of fDNA-based nanoprobes with light-activatable sensing and imaging functions, which provides precise control of recognition properties of fDNA with high spatiotemporal resolution. To explore the potential of organic NPs for biosensing applications, we have developed an enzyme-responsive fDNA-liposome as a universal sensing platform compatible with diverse biological targets as well as different detection methods including fluorescence, MRI, or temperature, making possible point-of-care diagnostics. To expand the application regime of organic NPs, we collaborated with the Zimmerman group to prepare single-chain block copolymer-based NPs and incorporated it with a variety of functions, including monovalent DNA for assembly, tunable surface chemistry for cellular imaging, and coordinative Cu(II) sites for catalyzing intracellular click reactions, demonstrating the potential of using organic NPs to create promising fDNA-NP systems with programmable functionalities. Furthermore, we survey our recent endeavor in integration of cell-specific aptamers with different NPs for targeted drug delivery, showing that introducing stimuli-responsive properties into NPs that target tumor microenvironments would enable safer and more effective therapy for cancers. Finally, current challenges and future perspectives in fDNA-mediated engineering of NPs for biomedical applications are discussed.
Topics: Animals; Biomedical Research; Biosensing Techniques; Colorimetry; DNA; Humans; Magnetic Resonance Imaging; Microscopy, Fluorescence; Nanoparticles
PubMed: 31411853
DOI: 10.1021/acs.accounts.9b00167 -
Chembiochem : a European Journal of... Apr 2024Due to nucleic acid's programmability, it is possible to realize DNA structures with computing functions, and thus a new generation of molecular computers is evolving to... (Review)
Review
Due to nucleic acid's programmability, it is possible to realize DNA structures with computing functions, and thus a new generation of molecular computers is evolving to solve biological and medical problems. Pioneered by Milan Stojanovic, Boolean DNA logic gates created the foundation for the development of DNA computers. Similar to electronic computers, the field is evolving towards integrating DNA logic gates and circuits by positioning them on substrates to increase circuit density and minimize gate distance and undesired crosstalk. In this minireview, we summarize recent developments in the integration of DNA logic gates into circuits localized on DNA substrates. This approach of all-DNA integrated circuits (DNA ICs) offers the advantages of biocompatibility, increased circuit response, increased circuit density, reduced unit concentration, facilitated circuit isolation, and facilitated cell uptake. DNA ICs can face similar challenges as their equivalent circuits operating in bulk solution (bulk circuits), and new physical challenges inherent in spatial localization. We discuss possible avenues to overcome these obstacles.
Topics: DNA; Logic; Computers, Molecular
PubMed: 38385968
DOI: 10.1002/cbic.202400080 -
Proceedings. Biological Sciences Aug 2021Ancient DNA (aDNA) has played a major role in our understanding of the past. Important advances in the sequencing and analysis of aDNA from a range of organisms have... (Review)
Review
Ancient DNA (aDNA) has played a major role in our understanding of the past. Important advances in the sequencing and analysis of aDNA from a range of organisms have enabled a detailed understanding of processes such as past demography, introgression, domestication, adaptation and speciation. However, to date and with the notable exception of microbiomes and sediments, most aDNA studies have focused on single taxa or taxonomic groups, making the study of changes at the community level challenging. This is rather surprising because current sequencing and analytical approaches allow us to obtain and analyse aDNA from multiple source materials. When combined, these data can enable the simultaneous study of multiple taxa through space and time, and could thus provide a more comprehensive understanding of ecosystem-wide changes. It is therefore timely to develop an integrative approach to aDNA studies by combining data from multiple taxa and substrates. In this review, we discuss the various applications, associated challenges and future prospects of such an approach.
Topics: DNA, Ancient; Ecosystem; Fossils; Geologic Sediments
PubMed: 34428961
DOI: 10.1098/rspb.2021.1252 -
Lab on a Chip May 2023A DNA hydrogel, owing to its dual properties of liquid and solid, is considered to be an ideal material for constructing biosensors that can integrate the advantages of...
A DNA hydrogel, owing to its dual properties of liquid and solid, is considered to be an ideal material for constructing biosensors that can integrate the advantages of both wet chemistry and dry chemistry. Nevertheless, it has struggled to cope with the demands of high-throughput analysis. A partitioned and chip-based DNA hydrogel is a potential avenue to achieve this, but currently remains a formidable challenge. Here, we developed a portable and partitioned DNA hydrogel chip that can be used for multitarget detection. The partitioned and surface-immobilized DNA hydrogel chip was formed by inter-crosslinking amplification by incorporating target-recognizing fluorescent aptamer hairpins into multiple rolling circle amplification products, which can achieve portable and simultaneous detection of multiple targets. This approach expands the application of semi-dry chemistry strategies, which can realize high throughput and point of care testing (POCT) of different targets, improving the development of hydrogel-based bioanalysis and providing new potential solutions for biomedical detection.
Topics: Hydrogels; DNA; Point-of-Care Testing; Aptamers, Nucleotide; Biosensing Techniques
PubMed: 37139578
DOI: 10.1039/d2lc01127a