دورية أكاديمية
In Vitro Characterization of Protein:Nucleic Acid Liquid-Liquid Phase Separation by Microscopy Methods and Nanoparticle Tracking Analysis.
| العنوان: | In Vitro Characterization of Protein:Nucleic Acid Liquid-Liquid Phase Separation by Microscopy Methods and Nanoparticle Tracking Analysis. |
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| المؤلفون: | do Amaral MJ; Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. marianajamaral@yahoo.com.br., Passos YM; Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil., Almeida MS; Protein Advanced Biochemistry, Institute of Medical Biochemistry Leopoldo de Meis and National Center for Structural Biology and Bioimaging, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil., Pinheiro AS; Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil., Cordeiro Y; Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. yraima@pharma.ufrj.br. |
| المصدر: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2551, pp. 605-631. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press, |
| مواضيع طبية MeSH: | Nucleic Acids* , Aptamers, Nucleotide* , Nanoparticles* , Intrinsically Disordered Proteins*/chemistry, Mice ; Animals ; Microscopy ; Recombinant Proteins |
| مستخلص: | Uncontrolled assembly/disassembly of physiologically formed liquid condensates is linked to irreversible aggregation. Hence, the quest for understanding protein-misfolding disease mechanism might lie in the studies of protein:nucleic acid coacervation. Several proteins with intrinsically disordered regions as well as nucleic acids undergo phase separation in the cellular context, and this process is key to physiological signaling and is related to pathologies. Phase separation is reproducible in vitro by mixing the target recombinant protein with specific nucleic acids at various stoichiometric ratios and then examined by microscopy and nanotracking methods presented herein. We describe protocols to qualitatively assess hallmarks of protein-rich condensates, characterize their structure using intrinsic and extrinsic dyes, quantify them, and analyze their morphology over time. Analysis by nanoparticle tracking provides information on the concentration and diameter of high-order protein oligomers formed in the presence of nucleic acid. Using the model protein (globular domain of recombinant murine PrP) and DNA aptamers (high-affinity oligonucleotides with 25 nucleotides in length), we provide examples of a systematic screening of liquid-liquid phase separation in vitro. (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.) |
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| فهرسة مساهمة: | Keywords: Amyloid fibrils; Biomolecular condensates; Coacervation; DNA aptamers; Differential interference contrast (DIC) microscopy; Fluorescence microscopy; Liquid-liquid phase separation (LLPS); Nanoparticle tracking analysis (NTA); Phase transitions; Prion protein (PrP); Transmission electron microscopy (TEM) |
| المشرفين على المادة: | 0 (Nucleic Acids) 0 (Aptamers, Nucleotide) 0 (Recombinant Proteins) 0 (Intrinsically Disordered Proteins) |
| تواريخ الأحداث: | Date Created: 20221031 Date Completed: 20221101 Latest Revision: 20221107 |
| رمز التحديث: | 20221213 |
| DOI: | 10.1007/978-1-0716-2597-2_37 |
| PMID: | 36310228 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1940-6029 |
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| DOI: | 10.1007/978-1-0716-2597-2_37 |