Visualization of NRAS RNA G-Quadruplex Structures in Cells with an Engineered Fluorogenic Hybridization Probe

Area of Science:

• Molecular Biology
• RNA Structure and Dynamics
• Biochemistry

Background:

• RNA G-quadruplexes are crucial secondary structures in guanine-rich sequences.
• In vitro studies dominate G-quadruplex folding research, limiting cellular insights.
• Accurate intracellular identification of RNA G-quadruplex formation remains challenging.

Purpose of the Study:

• To design and synthesize a novel probe for visualizing G-quadruplexes in the NRAS mRNA 5'-UTR.
• To develop a tool for direct and precise detection of specific RNA G-quadruplex structures.
• To establish a new method for investigating RNA G-quadruplex folding in cellular environments.

Main Methods:

• Design and synthesis of a G-quadruplex-triggered fluorogenic hybridization (GTFH) probe (ISCH-nras1).
• The probe incorporates a G-quadruplex-specific fluorescent light-up moiety and a DNA hybridization sequence.
• Evaluation of probe performance for NRAS RNA G-quadruplex detection in vitro and in cells.

Main Results:

• The ISCH-nras1 probe successfully visualized G-quadruplexes in the NRAS mRNA 5'-UTR.
• The probe demonstrated direct and precise detection of targeted RNA G-quadruplex structures.
• Effective G-quadruplex visualization was achieved both in vitro and within living cells.

Conclusions:

• The developed GTFH probe is a valuable tool for studying RNA G-quadruplex folding.
• This represents a novel approach for designing smart probes for RNA structure analysis.
• The probe facilitates direct investigation of G-quadruplex structures within specific RNA molecules.