Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell

Abstract

Background: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell.

Methodology/principal findings: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs.

Conclusions/significance: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

Figure 1. Structure and photophysical properties of ECHO probes.

(A) Molecular structure and photophysical properties of D₅₁₄ and D₆₄₀ ECHO probes. (B) Typical absorption and fluorescence spectra of ECHO probes. The spectra of a single-stranded anti -gau-D₅₁₄ (ss) and the hybrid with the complementary RNA (ds) were measured in a HEPES buffer (120 mM KCl, 5 mM NaCl, 25 mM HEPES, pH = 7.2). Emission spectra were obtained at 513 nm.

Figure 2. Imaging of expressed mRNA in living HeLa cells.

Mixtures of ECHO probe (10 µM) and fluorescent protein-encoding plasmid (50 ng/µL) in sterilized water were microinjected into a living HeLa cell. Images were acquired every 10 min for 9 h after microinjection, the acquisition times from the onset being displayed in each image (hh:mm). The probe fluorescence was collected with a yellow-green filter set (Ex 500/24–25, DM 520, Em 542/27–25) for anti -gau-D₅₁₄ and anti -aga-D₅₁₄ (A, C, I, and K) and a red filter set (Ex 575–625, DM 645, Em 660–710) for anti -ggc-D₆₄₀ (E and G). Fluorescence from fluorescent proteins was collected with an orange filter set (Ex 545/25, DM 570, Em 605/70) for HcRed1 and DsRed2-mito (B, D, F, and H) and a cyan filter set (Ex 436/25, DM 455, Em 480/40) for mTFP1-mito (J and L). Scale bar, 20 µm.

Figure 3. Fluorescent puncta in the nucleus of a HeLa cell.

Images were acquired at 1 h after microinjection of anti -aga-D₅₁₄ (10 µM) and pmTFP1-mito-Tag(aga) ×64 (50 ng/µL). Scale bar, 10 µm. (A–C) The cell nucleus including fluorescence-labeled PSP1. (A) Fluorescence from expressed mDsRed-PSP1, (B) fluorescence from the hybrid of anti -aga-D₅₁₄ and expressed mRNA, and (C) the merged image (inset, a magnified figure of one of the overlapping fluorescent puncta). (D–F) The cell nucleus including fluorescence-labeled SC35. (D) Fluorescence from expressed SC35-DsRed2, (E) fluorescence from the hybrid of anti -aga-D₅₁₄ and expressed mRNA, and (F) the merged image. (G–I) The cell nucleus including fluorescence-labeled PML. (G) Fluorescence from expressed mDsRed–PML, (H) fluorescence from the hybrid of anti -aga-D₅₁₄ and expressed mRNA, and (I) the merged image.

Figure 4. Simultaneous dual RNA imaging in a single HeLa cell.

(A) Microinjection of a mixture of anti -ggc-D₆₄₀, anti -aga-D₅₁₄, pDsRed2-mito-Tag(ggc) ×64, and pmTFP1-mito-Tag(aga) ×64. Filter sets were used as described in Figure 2. (B) Microinjection of a mixture of anti -ggc-D₆₄₀ and anti -aga-D₅₁₄. (C) Microinjection of a mixture of anti -ggc-D₆₄₀, anti -aga-D₅₁₄, and pDsRed2-mito-Tag(ggc) ×64. (D) Microinjection of a mixture of anti -ggc-D₆₄₀, anti -aga-D₅₁₄, and pmTFP1-mito-Tag(aga) ×64. Images were acquired every 10 min, the acquisition times being displayed in each image (hh:mm). Scale bar, 20 µm.