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

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.

Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1.

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.