Production of the anti-G-quadruplex antibody BG4 for efficient genome-wide analyses: From plasmid quality control to antibody validation.

Abstract

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that can form at guanine-rich sequences of DNA and RNA in every kingdom of life. At the DNA level, G4s can form throughout genomes but they are prevalently found in promoter regions and at telomeres, and they have been attributed functions spanning from transcriptional regulation, to control of DNA replication, to maintenance of chromosome ends. Our understanding of the functions of G4s in cells has greatly improved with the development of specific anti-G4 antibodies, which allow the visualization of G4s by immunofluorescence but also the mapping of these secondary DNA structures genome wide. Whole genome identification of the location and abundance of G4s with techniques such as Chromatin Immunoprecipitation coupled with sequencing (ChIP-Seq) and Cleavage Under Target and Tagmentation (CUT&Tag) has allowed the profiling of G4 distribution across distinct cell types and deepen the understanding of G4 functions, particularly in the regulation of transcription. Crucial for these types of genome-wide studies is the availability of an anti-G4 antibody preparation with high affinity and specificity. Here, we describe a protocol for the expression and purification of the anti-DNA G4 structure antibody (BG4) first developed by the Balasubramanian group, which has been proven to selectively recognize G4 structures both in vitro and within cells, and which has great applicability in high-throughput techniques. We provide a detailed, step-by-step protocol to obtain active BG4 starting from a commercially available expression plasmid. We also describe three different approaches to validate the activity of the BG4 preparation.