DNA–protein interaction studies: a historical and comparative analysis

Abstract

DNA–protein interactions are essential for several molecular and cellular mechanisms, such as transcription, transcriptional regulation, DNA modifications, among others. For many decades scientists tried to unravel how DNA links to proteins, forming complex and vital interactions. However, the high number of techniques developed for the study of these interactions made the choice of the appropriate technique a difficult task. This review intends to provide a historical context and compile the methods that describe DNA–protein interactions according to the purpose of each approach, summarise the respective advantages and disadvantages and give some examples of recent uses for each technique. The final aim of this work is to help in deciding which technique to perform according to the objectives and capacities of each research team. Considering the DNA–binding proteins characterisation, filter binding assay and EMSA are easy in vitro methods that rapidly identify nucleic acid-protein binding interactions. To find DNA-binding sites, DNA-footprinting is indeed an easier, faster and reliable approach, however, techniques involving base analogues and base-site selection are more precise. Concerning binding kinetics and affinities, filter binding assay and EMSA are useful and easy methods, although SPR and spectroscopy techniques are more sensitive. Finally, relatively to genome-wide studies, ChIP–seq is the desired method, given the coverage and resolution of the technique. In conclusion, although some experiments are easier and faster than others, when designing a DNA–protein interaction study several concerns should be taken and different techniques may need to be considered, since different methods confer different precisions and accuracies.

Protein–Nucleic Acid Interactions for RNA Polymerase II Elongation Factors by Molecular Dynamics Simulations

“…Xray crystallography and cryogenic electron microscopy (cryo-EM) provide structural information on protein−nucleic acid systems. In addition to the structural biology techniques, many other experimental techniques including Forster resonance energy transfer (FRET), 13,14 nuclear magnetic resonance (NMR), 15,16 mass spectrometry, 17,18 small-angle X-ray scattering (SAXS), 19 electrophoretic mobility shift assay (EMSA), 20−22 footprinting, 20,21 cross-linking, 21,23 and chromatin immunoprecipitation (ChIP) 20,21,24,25 focus on protein− nucleic acid interactions and their dynamics during the biological processes. In addition to these studies, computational approaches based on molecular dynamics (MD) simulations have provided important insights into the dynamics of protein−nucleic acid interactions at the atomistic details.…”

Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay

“…Optimal: The detected binding signal of target protein to DNA may be due to non-specific binding. Therefore, to rule out this possibility, poly [d(I-C)] or poly [d(A-T)] are often used as non-specific competitors for EMSA ( Buratowski and Chodosh, 2001 ; Ferraz et al., 2021 ). Herein, we also performed the exact same procedure as steps 3–9 above, except that 1 μg poly [d(I-C)] (Roche, Cat# 10108812001) was added to the above protein-DNA reaction mixtures listed in Table 6 .…”

Complexome Profiling—Exploring Mitochondrial Protein Complexes in Health and Disease

“…CP has not been popular for the analysis of DNA-interacting protein complexes. These have been addressed with more targeted approaches like chromatin immunoprecipitation (ChIP) ( Das et al, 2004 ), electrophoretic mobility shift assay (EMSA) ( Hellman and Fried, 2007 ) and a variety of Co-IP techniques ( Sahr and Buchrieser, 2013 ), which have been extensively reviewed by Ferraz et al (2021) . Although both genomic and mitochondrial DNA molecules that interact with proteins are too large to enter standard native gels and do not exist in populations separable by size, like RNPs, CP could be useful for decomposing DNA-associated protein complexes.…”