Poly(A)+ Sensing of Hybridization-Sensitive Fluorescent Oligonucleotide Probe Characterized by Fluorescence...

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Ribonucleic acid (RNA) is crucial for cellular processes, necessitating methods for direct visualization and quantification of its molecular dynamics in living cells.
• Understanding RNA metabolism requires tools to observe RNA behavior in real-time within the cellular environment.
• Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes offer low fluorescence background, making them suitable for sensitive RNA detection.

Purpose of the Study:

• To apply fluorescence correlation spectroscopy (FCS) with ECHO probes for visualizing and quantifying messenger RNA (mRNA) poly(A) tail dynamics in living cells.
• To demonstrate the capability of ECHO probes to detect and measure probe-target interactions, changes in fluorescence brightness, and diffusion coefficients upon binding.
• To validate the uptake and utility of ECHO probes for mRNA detection in HeLa cells.

Main Methods:

• Utilized exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes targeting the poly(A) tail of mRNA.
• Employed fluorescence correlation methods, specifically fluorescence correlation spectroscopy (FCS), for quantitative analysis.
• Performed experiments in living HeLa cells to demonstrate probe uptake and mRNA detection.

Main Results:

• Successfully visualized and quantified the interaction between ECHO probes and target mRNA.
• Demonstrated changes in fluorescence brightness and diffusion coefficient upon probe-target binding.
• Confirmed the uptake of ECHO probes in HeLa cells for effective mRNA detection.

Conclusions:

• Fluorescence correlation methods combined with ECHO probes provide a powerful tool for visualizing and quantifying intracellular mRNA dynamics.
• This approach offers new insights into RNA metabolism by enabling the study of probe-target interactions and molecular changes.
• The demonstrated utility in HeLa cells suggests broad applicability for studying mRNA in various cellular contexts.