Molekuliarnaia biologiia · 2016
M Ashwini、S B Murugan、S Balamurugan 等
"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.
Biotechnology Letters · 2003
Dong-Chuan Guo、D. Milewicz
暂无摘要
Heliyon · 2024
Yuxin Ma、Zhaoyang Zhang、Bin Jia 等
DNA synthesis and assembly primarily revolve around the innovation and refinement of tools that facilitate the creation of specific genes and the manipulation of entire genomes. This multifaceted process encompasses two fundamental steps: the synthesis of lengthy oligonucleotides and the seamless assembly of numerous DNA fragments. With the advent of automated pipetting workstations and integrated experimental equipment, a substantial portion of repetitive tasks in the field of synthetic biology can now be efficiently accomplished through integrated liquid handling workstations. This not only reduces the need for manual labor but also enhances overall efficiency. This review explores the ongoing advancements in the oligonucleotide synthesis platform, automated DNA assembly techniques, and biofoundries. The development of accurate and high-throughput DNA synthesis and assembly technologies presents both challenges and opportunities.
Angewandte Chemie (International ed. in English) · 2022
Eric Koesema、Animesh Roy、Nicholas G Paciaroni 等
There is considerable interest in the development of libraries of non-peptidic macrocycles as a source of ligands for difficult targets. We report here the solid-phase synthesis of a DNA-encoded library of several hundred thousand thioether-linked macrocycles. The library was designed to be highly diverse with respect to backbone scaffold diversity and to minimize the number of amide N-H bonds, which compromise cell permeability. The utility of the library as a source of protein ligands is demonstrated through the isolation of compounds that bind Streptavidin, a model target, with high affinity.
Methods in molecular biology · 2007
Dong-Chuan Guo、D. Milewicz
暂无摘要
Nanomedicine (London, England) · 2022
Bryant C Nelson、Sven Even Borgos
暂无摘要
Journal of visualized experiments : JoVE · 2023
Lukasz S Borowski、Karolina Kasztelan、Jolanta Czerwinska-Kostrzewska 等
The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.
Journal of the American Chemical Society · 2024
Jun-Feng Chen、Vittal Bhat、C. Hawker
The development of synthetic oligomers as discrete single molecular entities with accurate control over the number and nature of functional groups along the backbone has enabled a variety of new research opportunities. From fundamental studies of self-assembly in materials science to understanding efficacy and safety profiles in biology and pharmaceuticals, future directions are significantly impacted by the availability of discrete, multifunctional oligomers. However, the preparation of diverse libraries of discrete and stereospecific oligomers remains a significant challenge. We report a novel strategy for accelerating the synthesis and isolation of discrete oligomers in a high-throughput manner based on click chemistry and simplified bead-based purification. The resulting synthetic platform allows libraries of discrete polyether oligomers to be prepared and the impact of variables such as chain length, number, and nature of side chain functionalities and molecular dispersity on antibacterial behavior examined. Significantly, discrete oligomers were shown to exhibit enhanced activity with lower toxicity compared with traditional disperse samples. This work provides a practical and scalable methodology for nonexperts to prepare libraries of multifunctional discrete oligomers and demonstrates the advantages of discrete materials in biological applications.
International Journal of Applied Pharmaceutics · 2024
Koppula Jayanthi、Syed Suhaib Ahmed、Mohd Abdul Baqi 等
Objective: Thymidylate kinase (TMK) plays a crucial role in bacterial DNA synthesis by catalyzing the phosphorylation of deoxythymidine monophosphate (dTMP) to form deoxythymidine diphosphate (dTDP). Consequently, this enzyme emerges as a promising target for developing novel antibacterial drugs. However, no antibiotics were reported for this target, especially active against Staphylococcus aureus thymidylate kinase.
Methods: Benzylidene acetamide-based ligands were examined for their potency using the in silico method. These novel ligand structures were built using ChemDraw software. The protein was retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) website. The molecular docking and binding free energy calculation by prime Molecular Mechanics in Generalized Bond Surface Area (MM-GBSA) was performed for selected ligands. A 100 ns molecular dynamic simulation was also performed to assess the stability of the potential ligand as TMK inhibitors.
Results: All ten molecules have shown good glide scores and hydrophobic and hydrogen hydrophobic hydrogen bonding interactions with Arg48, Arg36, and π-π stacking Phe66 in the TMK enzyme (PDB: 4HLC). Among them, N-(2-ethylphenyl)-2-(4-((4-nitrobenzylidene) amino) phenoxy) acetamide molecule had high XP-docking scores of-3.27 kcal/mol based on extra-precision data. Prime Molecular Mechanics in Generalized Bond Surface Area study (MM-GBSA) studies also showed promising binding affinities that are ΔBind (-65.80), ΔLipo (-28.55), and ΔVdW (-55.10). Phe66 amino acid residue maintained continuous connections with the ligand during MD simulation. This ligand showed promising binding affinity with the SaTMK target.
Conclusion: The N-(2-ethylphenyl)-2-(4-((4-nitrobenzylidene) amino) phenoxy) acetamide ligand at the position of the benzene ring displayed nitrogen and oxygen group, thus indicating good potential activity as the inhibitor of TMK to treat antibacterial agents.
Trends in biotechnology · 2025
Weiyi Li、Po-Hsiang Hung、Takeshi Matsui 等
DNA can be engineered to produce new biologics, gene therapies, and cellular therapies, and to reprogram organisms. Having the ability to engineer DNA at scale can accelerate the development of these applications. Existing technologies excel at short oligonucleotide synthesis by chemical or enzymatic methods (up to 2000 bp) and intermediate-size DNA assembly (up to 5-7 kb). Yet synthesizing sequence-validated longer DNA (>10 kb) and/or constructing highly complex combinatorial DNA libraries at scale remains a significant challenge, due largely to technical and cost barriers. Inspired by recent studies on an in vivo DNA processing platform for megabase-long DNA assembly and on high-throughput sequence verification, we discuss how these platforms may be used to achieve DNA engineering at scale.
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