Development of Highly Fluorogenic Styrene Probes for Visualizing RNA in Live Cells

Abstract

Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the nucleolus and cytoplasm. However, the application of these indole-based dyes in cell imaging is limited by their moderate fluorescence enhancement and quantum yields, as well as relatively high background associated with these green-emitting dyes. In this work, we have investigated the positional and electronic effects of the electron donor by generating regioisomeric and isosteric analogues of the indole ring. Select probes exhibited large Stokes shifts, enhanced molar extinction coefficients, and bathochromic shifts in their absorption and fluorescence wavelengths. In particular, the indolizine analogues displayed high membrane permeability, strong fluorogenic responses upon binding RNA, compatibility with fluorescence lifetime imaging microscopy (FLIM), low cytotoxicity, and excellent photostability. These indolizine dyes not only give rise to rapid, sensitive, and intense staining of nucleoli in live cells but can also resolve subnucleolar structures enabling highly detailed studies of nucleolar morphology. Furthermore, our dyes can partition into RNA coacervates and resolve the formation of multiphase complex coacervate droplets. These indolizine-containing styrene probes offer the highest fluorescence enhancement among the RNA-selective dyes reported in the literature; thus, these new dyes are excellent alternatives to the commercially available RNA dye, SYTO RNASelect, for visualizing RNA in live cells and in vitro.

Figure 1

Chemical structures of the previously reported styrene-based RNA-selective dyes, MPI and IN, and the new dyes 1a–1d reported in the current study. The electron donor and acceptor moieties are highlighted in blue and red, respectively.

Scheme 1. Synthesis of the Styrene Dyes 1a–1d

Figure 2

Absorption and emission spectra of (a) 1c, (b) 1d, and (c) MPI in T.E. buffer (pH 7.5) or RNA (Type IV RNA from torula yeast; 200 μg/mL). Dye concentration: 10 μM. Dashed and solid lines indicate absorbance and emission, respectively. (d) Comparison of fluorescence intensity between MPI, 1c, and 1d in buffer vs dyes in RNA solution (200 μg/mL) dye concentration 10 μM. Dashed lines reflect the fluorescence intensity of unbound dye, and solid lines indicate the fluorescence intensity of dyes in RNA solution. (e) Fluorescence titration of 1c (10 μM) incubated with increasing concentration of nucleic acids or protein (BSA; bovine serum albumin).

Figure 3

Confocal fluorescence images of live HeLa cells incubated with 20 μM MPI (λ_ex = 470 nm, λ_em = 525–555 nm), 1c (λ_ex = 550 nm, λ_em = 580–620 nm), and 1d (λ_ex = 500 nm, λ_em = 560–600 nm) for 30 min. TPC: transmission phase contrast. Scale bar: 10 μm.

Figure 4

(a) Confocal fluorescence images of PFA-fixed HeLa cells incubated with 20 μM 1c and 1d for 30 min and 1 μg/mL Hoechst 33342 for 30 min. Scale bar: 10 μm. (b) Zoomed-in image of HeLa cells stained with 20 μM 1c for 30 min. Image shows the subnucleolar components FC (indicated by the white arrowhead), DFC, and GC (indicated by the white arrows). TPC: transmission phase contrast. Scale bar: 3 μm.

Figure 5

Comparison of intensity and FLIM phasor processed images of HeLa cells stained with 1c (20 μM). Separation of lifetimes was performed using FLIM phasor analysis, in which very short lifetimes appear on the bottom right and very long lifetimes appear on the bottom left. The phasor plot shows four unique lifetimes (color-coded on the FLIM image), corresponding to fluorophores within the nucleolus, nucleus, cytoplasm, and surrounding media. Scale bar: 10 μm.

Figure 6

Comparison of photobleaching of SYTO RNASelect, 1c, and 1d. Confocal fluorescence images of PFA-fixed HeLa cells incubated with 1c and 1d (1 μM) and SYTO RNASelect (1 μM). Scale bar: 20 μm.

Figure 7

Partitioning of 1c (10 μM) within the RNA/spermine condensates. The top row shows images enlarged by 1×, while the bottom row shows images enlarged by a factor of 8×. The left column shows images based on fluorescence intensity alone, and the middle column shows images resolved using FLIM. The phasor plot indicates the presence of a single lifetime species of 1c (2.6 ns). Scale bar: 10 μm.