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.
Highlights
Localizing ribonucleic acids (RNAs) and determining their intracellular dynamics are longstanding challenges in biochemistry and cell biology [1] and are critical for understanding a wide variety of cellular activities [2]
The results indicate that the application of the exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probe to an analysis of intracellular messenger RNA (mRNA)
A series of hybridizing ECHO probes different excitation wavelength (Dnnn) were synthesized by a conventional phosphoramidite method [36]
Summary
Localizing ribonucleic acids (RNAs) and determining their intracellular dynamics are longstanding challenges in biochemistry and cell biology [1] and are critical for understanding a wide variety of cellular activities [2]. There is significant interest in applying the high spatial and temporal resolution of optical microscopy to localizing and determining the dynamics of intracellular RNA. Determining the localization, expression, kinetics, and function of mRNA in live cells has significance for the understanding of gene expression and gene regulation. As mRNAs have no distinguishing features in unstained brightfield microscopy, several techniques have been developed for the in vitro imaging of mRNAs in fluorescent microscopes. Each of these methods has its strengths and drawbacks. Numerous derivative probes and improvements to the original MS2-GFP system have been proposed, including reporters with shorter and more configurable RNA binding domains [9,10], repeating strings of fluorescent proteins for increased brightness [10], and split protein complexes that fluoresce conditionally in the case of correct
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