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Antiviral Agents and Rational Drug Design
Published in Nathan Keighley, Miraculous Medicines and the Chemistry of Drug Design, 2020
In order to be able to identify targets against which drugs can be developed, knowledge of the structure and life cycle of viruses is paramount. A virus particle can simply be considered as a protein package that contains a type of nucleic acid with which it can infect host cells and hijack the host cell’s machinery to reproduce itself. Viruses contain one or more molecules of either RNA or DNA, but not both, thus are defined as either RNA viruses or DNA viruses. The RNA can be single-stranded (ssRNA) as is the case for most viruses, or double stranded, where the base sequence of the RNA that is the same as viral mRNA is called the (+) strand and its complementary partner is the (−) strand. Most DNA viruses contain the typical double stranded DNA, but single-stranded DNA is present in some viruses. There is great variation between viruses in the size of the nucleic acid, ranging from genomes coding for just three-to-four proteins to larger genomes that code for over one hundred proteins.
Disease Prediction and Drug Development
Published in Arvind Kumar Bansal, Javed Iqbal Khan, S. Kaisar Alam, Introduction to Computational Health Informatics, 2019
Arvind Kumar Bansal, Javed Iqbal Khan, S. Kaisar Alam
Many types of proteins bind to DNA to regulate the transcription or the repair of the DNA. For example, transcription-factors bind to DNA and transcription-proteins to regulate the DNA-transcription to mRNA; DNA-polymerase synthesizes the DNA molecules; helicases are used to unbind the DNA double-strand by breaking down the hydrogen bonds between nucleotides in opposite strands. Some proteins bind to double-stranded DNA, while others bind to single-stranded DNA. The protein-domains that recognize DNA-patterns to bind are called DNA-binding domains.
Mitochondrial Genome Damage, Dysfunction and Repair
Published in Shamim I. Ahmad, Handbook of Mitochondrial Dysfunction, 2019
Kalyan Mahapatra, Sayanti De, Sujit Roy
Mitochondrial genome of many eukaryotes had been considered to exist in the form of super-coiled circular DNA. However, there is now strong evidence that many of these circular mapped mtDNA consists primarily of linear, multimeric head-to-tail concatemers (Bendich, 1993, 1996). These, for example, have been found in few unrelated organisms such as ciliates, Apicomplexa (Plasmodium and its relatives), fungi, Chlorophycean green algae (Chlamydomonas and relatives) and several Cnidarian animals. These linear molecules contain various specialized end structures, such as covalently closed single-stranded DNA termini and terminally attached proteins. They also tend to have telomere-like repeats of differing lengths. In fact, the difference in size of mitochondrial genomes is mostly caused by the variations in the length and organization of intergenic regions which, in some cases are consisting of extensive tandem-repeats or stem-loop motifs.
Bioinformatic analysis of the expression profile and identification of RhoGDI2 as a biomarker in imatinib-resistant K562 cells
Published in Hematology, 2023
Yulin Yang, Fangmin Zhong, Junyao Jiang, Meiyong Li, Fangyi Yao, Jing Liu, Ying Cheng, Shuai Xu, Song Chen, Haibin Zhang, Yanmei Xu, Bo Huang
Total RNAs were extracted from IS-K562 and IR-K562 using TRNzol Universal reagent (TIANGEN, Beijing, China). NanoDrop 2000 (Thermo, Wilmington, U.S.A.) and Agilent Bioanalyzer 2100 (Agilent, California, U.S.A.) were used to determine the quantity and quality of RNA. Gene expression was analysed by GeneChip®PrimeView™ Human Gene Expression Array (Affymetrix, California, U.S.A.). Briefly, the RNA samples were amplified using the GeneChip 3′IVT Express Kit for Array Analysis. The total RNAs first underwent reverse transcription reaction to obtain first-strand cDNA. The single-stranded DNA then underwent second-strand cDNA synthesis and purification to become a double-stranded cDNA template. The template was amplified using the GeneChip 3′IVT Express Kit to obtain amplified RNA and biotin labeling. The aRNA was purified and then segmented and hybridized by GeneChip Hybridization Oven645 (Affymetrix, California, U.S.A.). After hybridization, the chip was washed and dyed (GeneChip Fluidics Station 450), and the images and raw data were scanned by the GeneChip Scanner 3000 (Affymetrix, California, U.S.A.). The process was as follows: First-Strand cDNA Synthesis 42°C, 2 h; Second-Strand cDNA Synthesis 16°C, 1 h, 65°C, 10 min; IVT 40°C, 16hr; Fragmentation 94°C, 35 min; Hybridization 98°C, 10 min, 45°C, 3 min.
Kinetic analysis of ternary and binary binding modes of the bispecific antibody emicizumab
Published in mAbs, 2023
Stefanie Mak, Agnes Marszal, Nena Matscheko, Ulrich Rant
The targets FIX and FX were stably immobilized on the sensor surface via DNA connectors. To this end, proteins were coupled to single-stranded DNA ligand strands 1 and 2, respectively. 100 µg of FX (HFIX 1009, Enzyme Research Laboratories) and FIX (HFX 1010, Enzyme Research Laboratories) were conjugated via amine-coupling (NHS EDC chemistry) to 48mer DNA ligand strands 1 and 2, respectively (Supplementary Figure S1 A), using the kits HK-NHS-1 for FX and HK-NHS-4 for FIX (Dynamic Biosensors GmbH). The resulting DNA-protein conjugates were separated from remaining free protein, free DNA, and potential protein-DNA multimers by ion-exchange chromatography on a proFIRE instrument (Dynamic Biosensors) (Supplementary Figure S1 B). The final yield of DNA-protein conjugates was 25 µg for FX and 20 µg for FIX, which was sufficient to functionalize the sensor surface more than 500 and 400 times, respectively (approximately 50 ng of protein consumption per functionalization). Pure DNA-protein conjugates were stored in buffer PE40 (10 mM NaH2PO4/Na2HPO4, pH 7.4, 40 mM NaCl, 50 μM EDTA, 50 μM EGTA and 0.05% Tween20) and frozen at −80°C until use.
Comet-FISH analysis of urothelial cells. A screening opportunity for bladder cancer?
Published in Expert Review of Molecular Diagnostics, 2023
Sebastiano La Maestra, Mirko Benvenuti, Francesco D’Agostini, Rosanna T. Micale
In the SCGE, DNA damage can be quantified using dyes that bind specifically to the chain of deoxyribonucleic acid. Ethyl bromide (EB) and 4,6-diamine-2-phenylindole (DAPI) are the most known and used dyes. EB intercalates into DNA, binding stably to double-stranded DNA. Differently, DAPI binds to the major groove. In particular, these dyes make bonds with single strands less potent than when binding to double-stranded DNA. Potentially, this should make the detection of single-stranded DNA fragments less efficient. On the other hand, tests carried out using orange acridine (AO) as a dye, capable of giving a yellow-green fluorescence to double-stranded DNA and red to single-stranded, compared to tests in which sample-stained EP did not report significant differences due to the possibility of marking single-stranded filament DNA [78].