Protein-DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

Abstract

The fields of application of functional proteomics are not limited to the study of protein-protein interactions; they also extend to those involving protein complexes that bind DNA or RNA. These interactions affect fundamental processes such as replication, transcription, and repair in the case of DNA, as well as transport, translation, splicing, and silencing in the case of RNA. Analytical or preparative experimental approaches, both in vivo and in vitro, have been developed to isolate and identify DNA/RNA binding proteins by exploiting the advantage of the affinity shown by these proteins toward a specific oligonucleotide sequence. The present review proposes an overview of the approaches most commonly employed in proteomics applications for the identification of nucleic acid-binding proteins, such as affinity purification (AP) protocols, EMSA, chromatin purification methods, and CRISPR-based chromatin affinity purification, which are generally associated with mass spectrometry methodologies for the unbiased protein identification.

Keywords: CRISPR-Cas9; ChIP; DNA−protein interactions; EMSA; RNA−protein interactions; mass spectrometry; proteomics; pull-down.

Figure 1

Schematic representation of DNA-pull-down assay workflow. (A) Probe design and synthesis. (B) Chemical or affinity immobilization of the probe on an insoluble solid support. (C) Incubation of nuclear extract with specific oligonucleotide for partners isolation. (D) Washings to remove unspecific proteins. (E) Elution of oligonucleotide interacting proteins. (F) Protein identification by mass spectrometry-based approaches.

Figure 2

Schematic representation of EMSA-MS experiment. (A) Nuclear proteins are incubated with an oligonucleotide probe and bands showing an electrophoretic mobility shift in comparison to the control are in situ hydrolyzed by trypsin and (B) proteins identified by LC-MS/MS approach. Here the control is the nuclear extract alone (second lane).

Figure 3

A generic representation of ChIP experiments. (A) After chromatin immunoprecipitation, (B) DNA–protein complexes are eluted. (C) For only DNA–protein complex isolation, ChIP-SICAP experiment is possible by DNA biotinylation and streptavidin purification, and then (D) elution and de-cross-linking. Finally, protein identification (E) was carried out by mass spectrometry approach (ChIP-MS) and DNA analysis (F), by (G) sequencing (ChIP-seq) or (H) by hybridization with a pull of fluorescence probe (ChIP on ChIP).

Figure 4

Schematic workflow of CRISPR-ChAP-MS experiment. (A) Overexpression of dCas9 and a specific RNA guide (gRNA), (B) binding to the specific promoter, cross-linking and sonication. (C) Immunoprecipitation of Cas9 for the isolation of DNA–protein complexes of the promoter of interest. After de-cross-linking, DNA (D) and protein (E) elution are carried out.