DNA–protein interactions: methods for detection and analysis

Abstract

DNA-binding proteins control various cellular processes such as recombination, replication and transcription. This review is aimed to summarize some of the most commonly used techniques to determine DNA-protein interactions. In vitro techniques such as footprinting assays, electrophoretic mobility shift assay, southwestern blotting, yeast one-hybrid assay, phage display and proximity ligation assay have been discussed. The highly versatile in vivo techniques such as chromatin immunoprecipitation and its variants, DNA adenine methyl transferase identification as well as 3C and chip-loop assay have also been summarized. In addition, some in silico tools have been reviewed to provide computational basis for determining DNA-protein interactions. Biophysical techniques like fluorescence resonance energy transfer (FRET) techniques, FRET-FLIM, circular dichroism, atomic force microscopy, nuclear magnetic resonance, surface plasmon resonance, etc. have also been highlighted.

Study of a humanized inhibitory anti-platelet glycoprotein VI phage antibody from a phage antibody library

“…Such 'immortalization' of antibody genes makes it technically feasible to produce antibodies rapidly in bacterial cultures and to manipulate their structures genetically so that antibodies to any antigen can be created. [23][24][25] Utilizing phage surface display technology, the VH and VL genes of human IgG could be amplified by reverse transcriptase polymerase chain reaction from the peripheral blood B lymphocytes of patients with ITP. A novel humanized genetically engineered anti-platelet GPVI ScFv antibody could then be constructed.…”

Computer-aided design of aptamers for cytochrome p450

“…This approach is similar to the Fragment-Based Drug Design method that based on selection (experimental or in silico) of small compounds able to interact with the target protein and linkage them in one molecule (Kumar et al, 2012;Joseph-McCarthy et al, 2014). Also short peptides or oligonucleotides frequently have been used instead of whole proteins or DNA/RNA molecules in protein-protein or protein-nuclear acids interactions studies (Veselovsky et al, 2007;Dey et al, 2012).…”

Probing and quantifying DNA–protein interactions with asymmetrical flow field-flow fractionation

“…[1,2] To achieve a better understanding of the nature of DNA-protein interaction, numerous research efforts have been devoted to measurement of binding kinetics and affinity, and identification of DNA sequences with affinity to transcription factors, polymerases, histones, etc. [3][4][5][6] In addition to endogenous interactions, synthetic DNA probes -DNA aptamers -that can interact with proteins and other biological targets strongly and specifically, have been discovered [7,8] with high utility in molecular detection and great potential as therapeutic agents. [9][10][11][12] Discovery of additional aptamers with high functionality and obtaining more knowledge on aptamer-target interaction are thus in great demand.…”

“…[3] Although high-throughput next generation sequencing techniques have greatly enhanced the discovery rate and reliability of DNA-protein interactions, [5,13] the first step of isolating the DNA-protein complex is limited to immunocapture on a solid support. Similar immobilization is also required for interaction studies using surface plasmon resonance [14] and protein arrays.…”