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.

scholarly journals Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind nucleic acids

2021 ◽  
Author(s):  
Luisa Gabriel ◽  
Bharath Srinivasan ◽  
Krzysztof Kuś ◽  
João F. Mata ◽  
Maria João Amorim ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Stress Granules ◽  
Helical Conformation ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Binding Ability ◽  
Amino Terminal ◽  
Mobility Shift Assay

Zα domains recognize the left-handed helical conformation of double stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 and DAI localize to the cytosolic stress granules mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the amino-terminal region of human DAI harbouring two Zα domains (ZαβDAI) and a nucleic acid-binding deficient mutant. Electrophoretic mobility shift assay demonstrated the ability of ZαβDAI to bind to hyperedited nucleic acids which are enriched in stress granules. Further, using immunofluorescence and immunoprecipitation coupled with mass-spectrometry, we identified several interacting partners of the ZαβDAI-RNA complex in-vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid binding ability or with RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to stress granules is mainly mediated by the nucleic acid binding ability of their Zα domains.

scholarly journals Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

2013 ◽  
Vol 4 (1) ◽  
pp. 3 ◽  
Author(s):  
Sarah E. Altschuler ◽  
Karen A. Lewis ◽  
Deborah S. Wuttke
Keyword(s):  
Nucleic Acid ◽  
Binding Affinities ◽  
Mobility Shift ◽  
Experimental Conditions ◽  
High Affinity ◽  
Apparent Binding Constant ◽  
Highly Sensitive ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, <em>Schizosaccharomyces pombe</em> Pot1 and <em>Saccharomyces cerevisiae </em>Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

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 An in silico approach to identification, categorization and prediction of nucleic acid binding proteins

2020 ◽  
Author(s):  
Lei Xu ◽  
Shanshan Jiang ◽  
Jin Wu ◽  
Quan Zou
Keyword(s):  
Nucleic Acid ◽  
Drug Targets ◽  
Nucleic Acid Binding ◽  
Structural Protein ◽  
Interaction Sites ◽  
Trained Classifier ◽  
Nucleic Acid Binding Proteins ◽  
Protein Nucleic Acid ◽  
And Function ◽  
Nucleic Acid Interactions

Abstract The interaction between proteins and nucleic acid plays an important role in many processes, such as transcription, translation and DNA repair. The mechanisms of related biological events can be understood by exploring the function of proteins in these interactions. The number of known protein sequences has increased rapidly in recent years, but the databases for describing the structure and function of protein have unfortunately grown quite slowly. Thus, improving such databases is meaningful for predicting protein–nucleic acid interactions. Furthermore, the mechanism of related biological events, such as viral infection or designing novel drug targets, can be further understood by understanding the function of proteins in these interactions. The information for each sequence, including its function and interaction sites, were collected and identified, and a database called PNIDB was built. The proteins in PNIDB were grouped into 27 classes, such as transcription, immune system, and structural protein, etc. The function of each protein was then predicted using a machine learning method. Using our method, the predictor was trained on labeled sequences, and then the function of a protein was predicted based on the trained classifier. The prediction accuracy achieved a score of 77.43% by 10-fold cross validation.

scholarly journals Viroplasm Protein P9-1 ofRice Black-Streaked Dwarf VirusPreferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

2013 ◽  
Vol 87 (23) ◽  
pp. 12885-12899 ◽  
Author(s):  
Jianyan Wu ◽  
Jia Li ◽  
Xiang Mao ◽  
Weiwu Wang ◽  
Zhaobang Cheng ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Binding ◽  
Structure And Function ◽  
Interior Structure ◽  
Mobility Shift ◽  
Chromatography Analysis ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Function

The P9-1 protein ofRice black-streaked dwarf virus(RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed forin vitrobinding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

Covalent Binding of Modified Nucleic Acids to Proteins as a Method for Investigation of Specific Protein—Nucleic Acid Interactions

ChemInform ◽  
2005 ◽  
Vol 36 (28) ◽  
Author(s):  
T. S. Zatsepin ◽  
N. G. Dolinnaya ◽  
E. A. Kubareva ◽  
M. G. Ivanovskaya ◽  
V. G. Metelev ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Covalent Binding ◽  
Specific Protein ◽  
Modified Nucleic Acids ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions
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