Plug-and-play fluorophores extend the spectral properties of Spinach

Abstract

Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. Spinach2 binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI), allowing the dynamic localizations of Spinach2-tagged RNAs to be imaged in live cells. The spectral properties of Spinach2 are limited by DFHBI, which produces fluorescence that is bluish-green and is not optimized for filters commonly used in fluorescence microscopes. Here we characterize the structural features that are required for fluorophore binding to Spinach2 and describe novel fluorophores that bind and are switched to a fluorescent state by Spinach2. These diverse Spinach2-fluorophore complexes exhibit fluorescence that is more compatible with existing microscopy filter sets and allows Spinach2-tagged constructs to be imaged with either GFP or YFP filter cubes. Thus, these "plug-and-play" fluorophores allow the spectral properties of Spinach2 to be altered on the basis of the specific spectral needs of the experiment.

Figure 1

Different fluorophores bind Spinach2 and form RNA–fluorophore complexes with unique spectral properties. Excitation (dotted line) and emission (solid line) spectra of Spinach2–fluorophore complexes. Excitation and emission spectra were collected in the presence of excess Spinach2 (5 μM) in the presence of different fluorophores (1 μM) in binding buffer (40 mM HEPES, pH 7.4, 125 mM KCl, 10 mM MgCl₂): (a) DFHBI, (b) DFHBI-1T, and (c) DFHBI-2T. Spectra are shown as the percent of the maximum excitation and emission fluorescence for each Spinach2–fluorophore complex. Excitation spectra were collected at the indicated emission maximum wavelength, and emission spectra were collected at the indicated excitation-maximum wavelength. The numbering in (a) indicates the atom numbering scheme for the imidazolone ring.

Figure 2

Live-cell imaging of Spinach2 fusion RNAs with different fluorophores. (a) COS7 cells expressing (CGG)₆₀-Spinach2 in the presence of either DFHBI or DFHBI-1T. Cells were initially cultured in the presence of 20 μM DFHBI (top panel), and images were acquired using a 100 ms exposure. The media was then exchanged with media containing 20 μM DFHBI-1T for 10 min, and images of the same cell nuclei were obtained using identical image acquisition conditions. Increased fluorescence was seen in cells cultured with DFHBI-1T (lower panel). (b) Quantification of average brightness of 10 foci normalized to brightness of DFHBI. Average and SEM values are shown. (c) Cells expressing (CGG)₆₀-Spinach2 in the presence of either DFHBI or DFHBI-2T were imaged using both GFP and YFP filter sets. Scale bar, 10 μm.