Recognition of nucleolin through interaction with RNA G-quadruplex

Area of Science:

• Biomarker Discovery
• Molecular Diagnostics
• Microfluidics

Background:

• Prostate cancer (PCa) diagnosis requires novel biomarkers for early detection.
• Nucleolin (NCL) is a potential PCa biomarker due to its surface expression on PCa cells and RNA-binding domains (RBDs).
• NCL's affinity for parallel G-quadruplexes (G4s) presents an opportunity for targeted detection.

Purpose of the Study:

• To develop a novel microfluidic platform for detecting Nucleolin (NCL) in biological samples.
• To utilize a specific RNA G4 (rG4) sequence from pre-microRNA 92b (pre-miR-92b) as a molecular recognition probe for NCL.
• To evaluate the potential of this platform as a biomedical tool for early prostate cancer diagnosis.

Main Methods:

• Development of a microfluidic platform for NCL detection.
• Utilized pre-miR-92b rG4 as a molecular probe, stabilized by acridine orange derivative C8.
• Employed FRET-melting assays, in silico studies, and fluorimetric titrations to characterize NCL-rG4 interactions and binding affinity (K_D 10^-12–10^-9 M).

Main Results:

• The pre-miR-92b rG4 structure, further stabilized by C8, demonstrated enhanced stability.
• Nucleolin RBD1,2 and C8 showed a synergistic effect on pre-miR-92b rG4 stabilization.
• High affinity binding of NCL RBD1,2 to both pre-miR-92b rG4 and the pre-miR-92b rG4/C8 complex was confirmed.
• The microfluidic platform successfully detected NCL in complex biological samples, including human plasma.

Conclusions:

• The developed microfluidic platform effectively detects Nucleolin (NCL) using a pre-miR-92b rG4 probe.
• The platform demonstrates high sensitivity and affinity for NCL detection in human plasma.
• This technology holds promise as a valuable biomedical tool for early prostate cancer diagnosis.