Visualization of RNA-Quadruplexes in Live Cells

Abstract

Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

Selective Luminescent Labeling of DNA and RNA Quadruplexes by π‐Extended Ruthenium Light‐Up Probes

“…As seen in Figure , incubating melanoma B16F10 cells with 4 (5 μ m ) for 15 min after MeOH fixation provided high‐quality images that highlight two main staining sites, the nucleoli and diffuse perinuclear cytoplasmic foci. This is reminiscent of what was observed with previously reported quadruplex‐selective smart probes, implying that 4 might primarily label RNA quadruplexes embedded in both nucleolar sites and cytoplasmic ribonucleoprotein assemblies in cells. Efforts must be invested to confirm the quadruplex nature of these cellular targets, but the quadruplex‐specificity of 4 shown above provides a great deal of confidence.…”

Templated Formation of Discrete RNA and DNA:RNA Hybrid G‐Quadruplexes and Their Interactions with Targeting Ligands

“…Strong arguments have been provided for the formation of both DNA and RNA G4 structures within cells with the use of G4‐specific antibodies and binding‐activated fluorescent G4‐targeting ligands . Several G4‐binding regulatory proteins have been identified, and G4 formation is now suspected to be involved in numerous pathogenic processes including degenerative disorders, oncogene regulation, and viral infections …”

A red-NIR fluorescent dye detecting nuclear DNA G-quadruplexes: in vitro analysis and cell imaging

“…For example, compounds that do not or poorly enter the nucleus are available only for cytoplasmic RNA G4 probing. 26,27 In addition, compounds must be selective for G4 nucleic acids. For example, Thioflavin T, 28 which also binds amyloid aggregates, cannot be used for in vivo imaging.…”

“…This indicates a remarkably different cellular entry and distribution of c-exNDI in comparison with recently described fluorogenic probes effectively targeting cellular G4s. 26,39 To characterize the nuclear localization of c-exNDI , treated cells were visualized using confocal microscopy. The compound appeared clustered in discrete foci all over the nucleus, with enhanced assemblage around and inside subnuclear organelles (Fig.…”