Visualizing stress granule dynamics with an RNA guanine quadruplex targeted ruthenium(ii) peptide conjugate

Abstract

Stress granules (SGs) are membraneless ribonucleoprotein assemblies that form in response to cellular stress. They have been linked to cell survival and cancer progression, though many questions remain regarding their structure, function and therapeutic potential. Live-cell fluorescence imaging is key to advancing understanding of SGs, but there are very few small-molecule probes reported that selectively image these organelles. RNA G-quadruplex (rG4) folding is believed to play a role in initiation of SG formation. Thus, to create a probe for SGs, we conjugated a G4 binding domain peptide from RNA helicase associated with AU-rich element (RHAU) to a luminescent [Ru(bpy)₂(PIC-COOH)]²⁺, Ru-RHAU. Ru-RHAU is designed to target rG4s and thus SGs in live cells. Studies in cellulo demonstrate that Ru-RHAU can induce SG formation in a concentration and time dependent manner and immunolabelling confirmed the complex remains associated with rG4s in the SGs. The SG stimulation is attributed to stabilization of rG4 by Ru-RHAU consistent with rG4's role in SG formation. Ru-RHAU shows low cytotoxicity under imaging conditions, facilitating prolonged observation in live cells. Interestingly, under more intense photoirradiation, Ru-RHAU induces phototoxicity through an apoptotic pathway. Ru-RHAU is a versatile tool for probing SG dynamics and function in cellular stress responses and has heretofore unconsidered potential in phototherapeutic applications targeting SGs.