Nucleic Acid-Binding Fluorochromes and Nanoparticles: Structural Aspects of Binding Affinity and Fluorescence Intensity

2018 ◽  
Vol 26 (2) ◽  
pp. 204-209
Author(s):  
Chul-Kyu Park ◽  
Sung Kil Hong ◽  
Yong Ho Kim ◽  
Hoonsung Cho
Keyword(s):  
Nucleic Acid ◽  
Binding Affinity ◽  
Fluorescence Intensity ◽  
Nucleic Acid Binding ◽  
Structural Aspects

scholarly journals mmCSM-NA: accurately predicting effects of single and multiple mutations on protein–nucleic acid binding affinity

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Thanh Binh Nguyen ◽  
Yoochan Myung ◽  
Alex G C de Sá ◽  
Douglas E V Pires ◽  
David B Ascher
Keyword(s):  
Nucleic Acid ◽  
Binding Affinity ◽  
Single Point ◽  
Point Mutations ◽  
Multiple Point ◽  
Missense Mutations ◽  
Nucleic Acid Binding ◽  
Single Point Mutations ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

Abstract While protein–nucleic acid interactions are pivotal for many crucial biological processes, limited experimental data has made the development of computational approaches to characterise these interactions a challenge. Consequently, most approaches to understand the effects of missense mutations on protein-nucleic acid affinity have focused on single-point mutations and have presented a limited performance on independent data sets. To overcome this, we have curated the largest dataset of experimentally measured effects of mutations on nucleic acid binding affinity to date, encompassing 856 single-point mutations and 141 multiple-point mutations across 155 experimentally solved complexes. This was used in combination with an optimized version of our graph-based signatures to develop mmCSM-NA (http://biosig.unimelb.edu.au/mmcsm_na), the first scalable method capable of quantitatively and accurately predicting the effects of multiple-point mutations on nucleic acid binding affinities. mmCSM-NA obtained a Pearson's correlation of up to 0.67 (RMSE of 1.06 Kcal/mol) on single-point mutations under cross-validation, and up to 0.65 on independent non-redundant datasets of multiple-point mutations (RMSE of 1.12 kcal/mol), outperforming similar tools. mmCSM-NA is freely available as an easy-to-use web-server and API. We believe it will be an invaluable tool to shed light on the role of mutations affecting protein–nucleic acid interactions in diseases.

scholarly journals Flow Cytometric Monitoring of Antibiotic-Induced Injury in Escherichia coli Using Cell-Impermeant Fluorescent Probes

2000 ◽  
Vol 44 (3) ◽  
pp. 676-681 ◽  
Author(s):  
Fiona C. Mortimer ◽  
David J. Mason ◽  
Vanya A. Gant
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Antimicrobial Agents ◽  
Flow Cytometric Analysis ◽  
Bacterial Cells ◽  
Nucleic Acid Binding ◽  
Permeabilized Cells ◽  
Sytox Green ◽  
Flow Cytometric

ABSTRACT Three fluorescent nucleic acid binding dyes—propidium iodide, TO-PRO-1, and SYTOX green—were evaluated, and their abilities to distinguish between bacterial cells with and without an intact cytoplasmic membrane were compared. Each dye was readily able to discriminate between healthy and permeabilized cells ofEscherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining-compromised, as opposed to healthy, cells in log-phase growth, than either PI or TO-PRO-1. Flow cytometric analysis of E. coli stained with these dyes after exposing them to several antimicrobial agents showed that all three dyes were able to detect antimicrobial action. Notably, however, the intensity of the cell-associated fluorescence was related to the mechanism of action of the antimicrobial agent. Large changes in fluorescence intensity were observed for all the dyes subsequent to β-lactam antibiotic action, but smaller changes (or no change) were seen subsequent to exposure to antimicrobials acting directly or indirectly on nucleic acid synthesis. Furthermore, cell-associated fluorescence did not relate to loss of viability as determined by plate counts. Despite offering much insight into antimicrobial mechanisms of action, these fundamental problems become relevant to the development of rapid antimicrobial susceptibility tests if colony formation is used as the standard.

scholarly journals Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 on its heterochromatin assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Atsuko Shirai ◽  
Takayuki Kawaguchi ◽  
Hideaki Shimojo ◽  
Daisuke Muramatsu ◽  
Mayumi Ishida-Yonetani ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Binding Affinity ◽  
Histone H3 ◽  
Pericentric Heterochromatin ◽  
Nucleic Acid Binding ◽  
Major Satellite ◽  
Satellite Rnas ◽  
Heterochromatin Assembly

SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1’s CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid–binding and H3K9me–binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H’s target specificity.

scholarly journals Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Zhe Zeng ◽  
Feng Deng ◽  
Ke Shi ◽  
Gang Ye ◽  
Gang Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Crystal Structures ◽  
Binding Affinity ◽  
Disulfide Bond ◽  
Rna Binding ◽  
Critical Role ◽  
Nucleic Acid Binding ◽  
Wild Type ◽  
Dimer Interface ◽  
Diarrhea Virus

ABSTRACTCoronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-ΔN7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-ΔN7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity.IMPORTANCECoronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

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