G4-Unfolder - PhpC
Molecular helicase (PhpC molecule) destabilizing G-quadruplexes.
Kit size : 1 or 5mg of PhpC
A molecule developed by Dr David Monchaud (Institut de Chimie Moléculaire de l'Université de Bourgogne, CNRS, France)
G-quadruplexes (G4s) are unusual DNA structures in which four guanines pair together in a planar arrangement. They are known to play roles various roles in gene regulation and expression. Tools are often used to label or stabilize these G-quadruplexes, but comparatively few tools exist to unwind these structures.
G4-unfolder is a small molecule that destabilizes G-quadruplexes.
Universal
G4-Unfolder works on DNA and RNA G4s
Physiologically relevant
G4-Unfolder does not strongly bind to G4 : it does not prevent enzyme binding
Widely validated
G4-Unfolder effects have been validated at the molecular, enzymatic and cellular levels.
Overview of G4-Unfolder effects
- Help enzymes go through G4 roadblocks, including Taq polymerase (qPCR stop assay) and Pif1 helicase (G4-helicase assay).
- Modulates the G4 landscape in human cells, shown by
- optical imaging in breast, lung and cervical cancer cells
- affinity precipitation in breast and colon cancer cells
- both RNAseq and proteomics in patient-derived astrocytes
- Functional consequences of PhpC destabilization were studied as a way to rescue
- the G4-associated genetic instability in cancer cells,
- the X-linked dystonia parkinsonism (XDP) phenotype in neural progenitor cells
- the maturation of G4-containing miRNA in cancer cells
Technical information on G4-unfolder
Molecular weight : 470.4 g/mol (PhpC.TFA)
Formula : C20H21F3N4O6 (with TFA)
Solubility : water
Appearance : solid (powder)
Enhancement of Pif1-mediated G4 unfolding in presence of G4-Unfolder (PhpC)
The presence of PhpC enhances the Pif1-mediated G4 unfolding (by 1.5- to 2.4-fold). TMPyP4, another molecule that supposedly destabilizes G4, decreases the unfolding efficiency (0.6-fold). Reference : Mitteau et al 2021 (https://doi.org/10.1021/jacs.1c04426)
Unfolding of pre-miRNA G4 with G4-Unfolder (PhpC) in lung cancer cells
Identifying and modulating G4 structure in pre-miR-3196 to restore normal lung cancer levels, offering a potential therapeutic strategy. In the result below, after incubation with phpc, the G4 amount dropped significantly (by 77%), indicating that PhPC unfolds r-G4-3196 in A549 cells.
(A) Representative image of A549 lung cancer cells transfected with FAM-RG4-3196, a G4-forming RNA sequence (visible in the green channel), incubated with PhPC (50µM, 24h), and subsequently labelled with QUMA-1, a fluorescent probe recognizing G4 (in red), and Hoescht, labelling nuclei (in blue).
(B) Integrated fluorescence values per cell emitted from the QUMA-1 (red signal, labelling all G4s) with or without incubation with PhPC.
(C) Colocalization coefficients between the rG4-3196 sequence and QUMA-1 probe, with and without incubation with PhPC.
Reference : Alexandre et al. 2025 (https://doi.org/10.1016/j.ijbiomac.2025.145263)
Modulation of RNA expression by G4-Unfolder (PhpC) in colorectal cancer cell line
Results of the G4RP-RT-qPCR performed with HT-29 cells either untreated or treated with PhpC (90 μM). Incubation of cells with PhpC triggered a significant decrease in UXT-AS1 and NRAS expression. Reference : Sharma et al 2025 (https://doi.org/10.1186/s12915-025-02322-8)
G4-Unfolder (PhpC) increases amplification speed of G4 strand in qPCR stop assay
qPCR stop assay relies on the amplification of a DNA fragment comprising a G4-forming sequence on one strand. The presence of a folded G4 acts as a roadblock to Taq, which stalls replication and leads to a decreased amplification efficiency. Here, in presence of PhPC, amplification of the G4 strand is faster, and speed of amplification on the non-G4-strand is not modified. Reference : Mitteau et al 2021 (https://doi.org/10.1021/jacs.1c04426)
Reduction of G4 circular dichroism signature of hTelo incubated with G4-Unfolder (PhpC)
Circular dichroism (CD) titrations were performed using the human telomeric G4-forming sequence (hTelo) and increasing amounts of PhpC (1−10 mol equiv). PhpC triggers a decrease (−17.5% at 10 mol equiv) of the CD signal of the G4 (collected at its maximum, 293 nm). Reference : Mitteau et al 2021 (https://doi.org/10.1021/jacs.1c04426)
Evaluation of G4-Unfolder (PhpC) toxicity in a cellular context
Vitality assay (MTT) in HeLa cells treated with PhpC at different concentrations for 24h. Reference : De Magis et al. 2023 (https://doi.org/10.1038/s41467-023-42494-x)
Cytotoxicity of PhpC and BRACO-19 in HT-29 cells using Sulforhodamine B (SRB) dye. Cells are treated with PhpC (0 – 100 μM) for 72 hours. IC50 of PhpC is established at 242µM. Reference : Sharma et al. 2024 (https://doi.org/10.1186/s12915-025-02322-8)