Design, synthesis, and evaluation of fluorogenic cyanine dyes for nucleolar RNA imaging in living cells: effect of regioisomers on probe functions

概要

The thesis consists of 5 chapters.
Introduction (chapter 1）
RNA-selective small molecular weight probes have been of great interest as a tool for the regulation,
inhibition, and/or study of biological RNA functions. Of my particular interest is the design of
fluorogenic dyes for the imaging of nucleolar RNA in living cells, given the considerable attention on the
role of nucleolus in major physiological functions such as stress response, development and aging, and
canceration. While the signaling ability of a commercially available dye (SYTO RNA-select) is
promising, it is not applicable to living cells, having a limited use for fixed and permeabilized cells. A
variety of chemical scaffolds have therefore been proposed for the imaging of nucleolar RNA in living
cells, but the signaling abilities of most probes are only moderate compared to SYTO RNA select. On the
other hand, our group has recently developed novel cyanine dyes, including red-emissive BIQ and
yellow-emissive BIOP. Both probes are characterized as outstanding off-on signaling ability and inherent
RNA selectivity over DNA in solutions and live cells, indicating that cyanine dyes are good candidates as
RNA selective dye for nucleolar RNA imaging in living cells.
In this work, three kinds of typical cyanine dyes and their regioisomers were examined for the
development of novel RNA selective dyes for nucleolar RNA imaging in living cells. In particular, I
examined effect of regioisomers on probe functions.
Results and discussions
Thiazole orange and their derivatives (chapter 2) : First, I focused on thiazole orange (TO, em = 532
nm), a typical unsymmetrical monomethine cyanine dye, due to its large fluorescence enhancement when
binding with nucleic acid as well as the easily modified structure. In addition, three kinds of TO
derivatives, TO-H, TO-C4, and 2TO were prepared, and their binding and signaling properties were
evaluated. It was found that TO showed the response selectivity for RNA over DNA (IRNA/IDNA = 2), and
its light up property (I/I0 = 580-fold, bound/free = 400) even compared with commercial SYTO RNA
select (I/I0 = 500-fold, bound/free = 16). More interestingly, 2TO (em = 532 nm), a regioisomer of TO,
did work much better as a RNA selective dye (IRNA/IDNA = 3.7) where the value of bound/free reached
1200 (I/I0 = 430-fold). Furthermore, it was found that TO had a good cell plasma and nuclear membrane
permeability in living cells, and was applicable to the imaging of nucleolar RNA in living cells.
Significantly, much better image was obtained by 2TO, where the nucleolus was clearly stained with a
much higher contrast, revealing the potential of 2TO as RNA-selective dye for live cell imaging. These
results may indicate that the isomerization of TO is key to improving RNA selectivity.
Quinoline blue and its regioisomer (chapter 3) : Based on the above-mentioned finding, quinoline blue
(QB, em = 609.5 nm), the first synthesized cyanine dye, as well as its regioisomer, 2QB (also known as
QV, em = 578 nm) were developed and examined as RNA selective dyes. It was found that both probes
showed RNA selectivity over DNA in solutions (QB, IRNA/IDNA = 1.6; 2QB, IRNA/IDNA = 2.8), and
applicable to nucleolar RNA imaging in living cells. ...