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

Abstract

Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA.

Keywords

• exciton-controlled hybridization-sensitive oligonucleotide probe
• fluorescence correlation spectroscopy
• dual-color fluorescence cross-correlation spectroscopy
• poly(A) tail
• mRNA