Brief Introduction to Nucleic Acid-Protein Interactions
|16.7.2020||Posted by tactical33 under Advertising & Marketing|
Proteins and nucleic acids are the main biological macromolecules that make up life, and studying the interaction between proteins and nucleic acids and the interaction between proteins and proteins is one of the important research fields in the post-genomic era. At present, there are many methods to study the interaction between proteins and nucleic acids. This article will introduce several of the main approaches:
Electrophoretic mobility shift assay (EMSA, also known as gel retardation assay or gel shift assay) is an in vitro method that detects interactions between proteins and nucleotides. It was originally used to detect transcription factors and is now further developed to study DNA-protein interactions, RNA-protein interactions, and even DNA-RNA interactions.
DNase I Footprinting Assay
This method uses DNase I to digest radio labeled or fluorescently labeled DNA fragments, followed by detection of the resulting cleavage patterns using gel electrophoresis. DNA cleavage patterns in the absence of DNA-binding proteins, commonly referred to as free DNA, were compared to those in the presence of DNA-binding proteins. If the protein binds to the DNA fragment, the binding site is protected from enzymatic cleavage. This protection will create a transparent area on the gel called a “footprint”. By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated from the minimum protein concentration at which the footprint is observed.
Chromatin immunoprecipitation (ChIP) analysis is a powerful method to study DNA-protein interactions. Based on in vivo technical design and the specificity of antigen-antibody interactions, it can reflect the physiological binding state between proteins and chromosomal DNA.
ChIP is often used to study transcription factor binding sites or protein modification sites. With the development of NGS, the combination of CHIP and NGS, namely chromatin immunoprecipitation sequencing (ChIP-Seq), now enables efficient and accurate screening and identification of protein binding sites throughout the chromosome. Briefly, in vivo protein-DNA interaction complexes were immobilized and digested into small fragments, which were then immunoprecipitated with antibodies against DNA-binding proteins. In this way, protein-bound DNA fragments are enriched and can be analyzed by NGS, so that protein-binding sites throughout the genome can be located and characterized. ChIP-Seq is a valuable tool for identifying and quantifying specific DNA sequences for protein binding or the presence of epigenetic modifications.
CLIP (cross-linked immunoprecipitation) is a method used to analyze RNA interactions with proteins or to localize precise RNA modifications. This technique is conducted by performing immunoprecipitation with antibodies against the target protein after UV cross-linking of the RNA-protein complex. CLIP-Seq, also known as HITS-CLIP, combines CLIP with high-throughput sequencing for the identification of binding sites on RBPs. CLIP-Seq can be used to map RNA binding of interacting proteins or RNA modification sites on a genome-wide scale, which helps to facilitate the understanding of post-transcriptional regulatory networks and mechanisms.
Compared with conventional methods and technology, RIP-ChIP/Seq has become increasingly more favored by the researchers because of its own a variety of advantages, including: high-throughput, applicable in any kinds of eukaryotic cells and tissues and simultaneously identify multiple RBPs of interest.