Fluorescent light-up aptamers (FLAPs) have emerged as valuable tools to visualize RNAs, but are mostly limited by their poor brightness, low photostability, and high fluorescence background in live cells. Exploiting the avidity concept, here we present two of the brightest FLAPs with the strongest aptamer–dye interaction, high fluorogenicity, and remarkable photostability. They consist of dimeric fluorophore-binding aptamers (biRhoBAST and biSiRA) embedded in an RNA scaffold and their bivalent fluorophore ligands (bivalent tetramethylrhodamine TMR2 and silicon rhodamine SiR2). Red fluorescent biRhoBAST–TMR2 and near-infrared fluorescent biSiRA–SiR2 are orthogonal to each other, facilitating simultaneous visualization of two different RNA species in live cells. One copy of biRhoBAST allows for simple and robust mRNA imaging with strikingly higher signal-to-background ratios than other FLAPs. Moreover, eight biRhoBAST repeats enable single-molecule mRNA imaging and tracking with minimal perturbation of their localization, translation, and degradation, demonstrating the potential of avidity-enhanced FLAPs for imaging RNA dynamics.
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M.S. and A.J. were supported by the Deutsche Forschungsgemeinschaft (DFG grant no. Ja794/11). We thank the Nikon Imaging Center, Heidelberg for granting access to their facilities and U. Engel for technical advice in fluorescence microscopy.
The authors declare no competing interests.
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Extended Data Fig. 1 Avidity dramatically enhances the binding strength between the bivalent TMR dyes (1 and 2) and dimeric RhoBAST aptamers.
a–e, Predicted secondary structures of RhoBAST (a); its dimeric variant with a 10-nucleotide linear and flexible linker (b); biRhoBAST with a rigid linker (a four-way junction with a stabilizing stem loop48) between aptamers (c). d,e, Dissociation constant (KD) measurements between RhoBAST variants (monomeric in blue, dimeric with a flexible linker in green, and biRhoBAST with a rigid linker in red) and bivalent TMR dye 1 (d); bivalent TMR dye 2 (e). Plots were obtained by titrating dye solutions with increasing concentrations of aptamers and recording the fluorescence intensities in ASBT. The final concentrations of the dyes (1 and 2) during measurements were 5 nM, 1 nM and 0.1 nM when RhoBAST, dimeric RhoBAST with a flexible linker and biRhoBAST were used as aptamers, respectively. KDavidity depends significantly on the rigidity, geometry and preorganization of the aptamers as well as on the linker length of the dyes, which also determines the strength of the H-dimers32,36.
Extended Data Fig. 2 Bivalent TMR dye TMR2 is superior to 1 and 2 for RNA imaging in live cells.
a, Live HEK293T cells expressing circular biRhoBAST or the control aptamer (Broccoli)37 were incubated with 50 nM of 1, 2 or TMR2 and imaged using a point-scanning confocal microscope. Two different contrast settings were shown for cells incubated with 1 and 2. Scale bars, 10 µm. b, The cytosolic fluorescence intensity (mean ± s.d.) of cells (N = 55, 52, 55, 55, 54, 60 cells from left to right) described in panel a. Although cells expressing biRhoBAST showed significantly higher fluorescence than control cells for all three dyes, TMR2 yielded a much higher signal than 1 and 2. P values were calculated using two-sided two-sample t-tests. c, HEK293T cells expressing circular biRhoBAST or the control aptamer (Broccoli)37 were fixed with paraformaldehyde, permeabilized, incubated with 50 nM of 1, 2 or TMR2 and imaged using a point-scanning confocal microscope. Scale bars, 10 µm. d, The cytosolic fluorescence intensity (mean ± s.d.) of the permeabilized cells (N = 68, 75, 79, 67, 72, 73 cells from left to right) described in panel c. All dyes yielded similar signals in fixed cells. P values were calculated using two-sided two-sample t-tests.
Extended Data Fig. 3 Evaluation of the orthogonality of biRhoBAST–TMR2 and biSiRA–SiR2 in vitro.
a, The fluorescence intensity change while titrating TMR2 (0.1 nM) with increasing concentrations of biRhoBAST or biSiRA in ASBT. While biRhoBAST yielded a dissociation constant of 40 pM with a ~40-fold fluorescence increase under these conditions, biSiRA yielded a dissociation constant of 6.0 nM with only ~5-fold fluorescence turn-on. Since monomeric SiRA has been shown to bind TMR with a KD of ~2 µM, this result were not unexpected29. b, The fluorescence intensity change while titrating SiR2 (0.1 nM) with increasing concentrations of biSiRA or biRhoBAST in ASBT. While biSiRA yielded a dissociation constant of 291 pM with a ~20-fold fluorescence increase under these conditions, biRhoBAST did not result in any fluorescence change. c, The fluorescence turn-on values (mean ± s.d.) for different combinations of biSiRA and biRhoBAST aptamers (500 nM) with SiR2 and TMR2 dyes (20 nM) of three independent measurements. The fluorescence values of the only dye solutions were normalized to 1.
Extended Data Fig. 4 TMR2 performs better than TMR-DN for imaging low abundant RNAs in live cells owing to its low background fluorescence.
a, Live HEK293T cells expressing circular biRhoBAST or Broccoli (control) were incubated with either TMR2 (50 nM) or TMR-DN (100 nM)16 and imaged. While both probes enabled imaging circular biRhoBAST with remarkable signal-to-background ratios, TMR2 showed lower background fluorescence in control cells expressing Broccoli aptamer. Scale bars, 10 µm. b, The fluorescence intensity (mean ± s.d.) in the cytosolic region of cells (N = 55, 52, 58, 58 cells from left to right) described in panel a using two-sided two-sample t-tests. c, Live HEK293T cells expressing mEGFP-biRhoBAST or mEGFP mRNA were incubated with either TMR2 (50 nM) or TMR-DN (100 nM) and imaged. Since the expression level of mEGFP-biRhoBAST mRNA is significantly lower than that of circular biRhoBAST, higher laser power was used in this experiment. TMR2 clearly enabled specific labeling of mEGFP-biRhoBAST mRNA in live cells with a signal-to-background ratio of 3. Whereas, TMR-DN did not yield a sufficient signal-to-background ratio to image mEGFP-biRhoBAST mRNA in this condition mainly due to the high background fluorescence. Scale bars, 10 µm. d, The fluorescence intensity (mean ± s.d.) in the cytosolic region of cells (N = 78 cells for all conditions) described in panel c. P values were calculated using two-sided two-sample t-tests.
Extended Data Fig. 5 One copy of biRhoBAST allows imaging tRNA, 7SK snRNA, and U6 snRNA and reports their localizations accurately.
a, HEK293T cells transfected with the stand-alone biRhoBAST construct (that uses the U6 promoter) or the empty vector (negative control) as well as mEGFP plasmid (transfection control) were fixed, incubated with 50 nM TMR2 and imaged on a scanning confocal microscope. Scale bars, 5 µm. b, HEK293T cells expressing tRNA-biRhoBAST or tRNA (negative control) from the U6 promoter as well as mEGFP (transfection control) were fixed, incubated with TMR2 (50 nM) and imaged with a scanning confocal microscope. TMR signal showed the expected cytosolic localization of the tRNA-biRhoBAST23,38,58. Scale bars, 10 µm. c, HEK293T cells expressing biRhoBAST-7SK or 7SK snRNA (negative control) from the U6 promoter as well as SC35-GFP protein (SC35 localizes in nuclear speckles) were fixed, incubated with TMR2 (50 nM) and imaged with a scanning confocal microscope. Transfected cells showed co-localized signals of GFP and TMR2 in nuclear speckles where 7SK RNA is expected to localize19,23. Scale bars, 5 µm. d, HEK293T cells expressing biRhoBAST-U6 or U6 snRNA (negative control) from the U6 promoter were fixed and incubated with a primary antibody targeting Cajal bodies (anti-Coilin) where U6 RNA was expected to accumulate23,39,40. Cells were stained with an Alexa488-conjugated secondary antibody and TMR2 (50 nM) imaged with a scanning confocal microscope. The TMR signal indicated the expected nuclear and Cajal body localization of the biRhoBAST-U6 RNA. Scale bars, 5 µm. e–h, The fluorescence intensity profiles of white lines shown in the merge images in panels a–d in green (GFP/Alexa488), red (TMR2) and blue (Hoechst) channels. These intensity profiles displayed; the nuclear localization of biRhoBAST (e), the primarily cytosolic localization of tRNA-biRhoBAST (f), localization of biRhoBAST-7SK to nuclear speckles is apparent from the colocalization of TMR2 and SC35 (g) and localization of biRhoBAST-U6 to Cajal bodies is apparent from the co-localization of TMR2 and anti-Coilin signals (h). i, Northern blot analysis of total RNAs isolated from cells expressing circular biRhoBAST, tRNA-biRhoBAST, biRhoBAST, biRhoBAST-7SK and biRhoBAST-U6. Due to the varying abundance of these transcripts, different amounts of total RNAs isolated from the sample group were loaded into each well to detect all of them at once. For lanes 5 and 6: 15 µg of total RNA; for lane 7: 10 µg of total RNA; for lanes 1, 2, and 3: 0.2 µg, 0.7 µg and 1.5 µg of total RNAs supplemented with control total RNA isolated from un-transfected cells (total amount of RNA per well ≈ 10 µg) to have comparable electrophoretic mobility among different sample groups. Expected lengths of untagged snRNAs and snRNAs tagged with one biRhoBAST unit was shown in the table. Total RNAs were isolated from the cells 48 h after the transfection, separated on a 1.2% agarose gel containing 0.4 M formaldehyde, transferred to a membrane, and UV-cross-linked. The membrane was labeled with a radioactively labeled biRhoBAST-specific DNA probe to show the different snRNA-biRhoBAST and potential biRhoBAST decay fragments. While for tRNA-biRhoBAST (2) no decay fragments were detected, we found that the major bands (full-length) of biRhoBAST-U6 (5) and biRhoBAST-7SK (6) represent 88 ± 2% and 80 ± 2% of all transcripts containing biRhoBAST, indicating a small fraction of biRhoBAST decay fragments. Two independent experiments were carried out with similar results.
Extended Data Fig. 6 One copy of biRhoBAST allows imaging a variety of mRNAs and reports their localizations accurately.
a, Live HEK293T cells expressing mAzurite-biRhoBAST, mAzurite-biRhoBAST-ARE or mAzurite mRNAs from a CMV promoter were visualized in the presence of 50 nM TMR2 on a scanning confocal microscope. AREs (AU-rich element) are known to reduce the half-life of mRNAs9,59,60. Scale bars, 5 µm. b, Live HEK293T cells expressing Kif18b-biRhoBAST or Kif18b mRNAs from a CMV promoter were visualized in the presence of 50 nM TMR2 on a scanning confocal microscope. Cells were also co-transfected with mAzurite to find the transfected cells. Scale bars, 5 µm. c, Live HEK293T cells expressing GAPDH-biRhoBAST or GAPDH mRNAs from a CMV promoter were visualized in the presence of 50 nM TMR2 on a scanning confocal microscope. Cells were also co-transfected with mAzurite to find the transfected cells. Scale bars, 5 µm. d, Live HEK293T cells expressing TNFα-biRhoBAST or TNFα mRNAs from a CMV promoter were visualized in the presence of 50 nM TMR2 on a scanning confocal microscope. Cells were also co-transfected with mAzurite to find the transfected cells. Scale bars, 5 µm. e, Live HEK293T cells expressing GFP-β-actin-biRhoBAST or GFP-β-actin mRNAs from the native chicken-β-actin promoter were visualized in the presence of 50 nM TMR2 on a scanning confocal microscope. Scale bars, 5 µm. f, Northern blot analysis of total RNAs isolated from cells expressing mAzurite-biRhoBAST, mAzurite-biRhoBAST-ARE, GFP-β-actin-biRhoBAST, GAPDH-biRhoBAST, TNFα-biRhoBAST or Kif18b-biRhoBAST mRNAs. Due to the varying abundance of these transcripts, different amounts of total RNAs isolated from the sample group were loaded into each well to detect all of them at once. For lanes 6, 8, 9 and 10: 15 µg of total RNA; for lane 2: 10 µg of total RNA; for lanes 3 and 4: 5 µg of total RNAs supplemented with control total RNA isolated from un-transfected cells (total amount of RNA per well ≈ 10 µg) to have comparable electrophoretic mobility among different sample groups. Expected size of the full-length mRNAs, biRhoBAST fragment and the 3’UTR are shown above. Total RNAs were isolated from the cells 48 h after the transfection, separated on a 1.2% agarose gel containing 0.4 M formaldehyde, transferred to a membrane, and UV-cross-linked. The membrane was labeled with a radioactively labeled biRhoBAST-specific DNA probe to show the different mRNA-biRhoBAST and potential biRhoBAST decay fragments. For all tested mRNAs the full-length transcripts were detected as major bands (>98%), proving that the biRhoBAST sequence does not cause accumulation of 3′ mRNA decay fragments in significant amounts. For that reason, the minimal decay products of biRhoBAST makes it an excellent tag to study mRNAs with different half-lives and lengths in living cells. Two independent experiments were carried out with similar results.
Extended Data Fig. 7 biRhoBAST–TMR2 can be used to detect specific single-molecule mRNA.
a,b, Schematic illustration of mAzurite mRNA tagged with either eight biRhoBAST units (a) or 24 MS2 stem loops (b). c, Spinning disk confocal images of live Cos7 cells expressing low levels of MCP-GFP and mAzurite-biRhoBAST8 or mAzurite-MS224 in the presence of 500 nM TMR2. Single mRNA molecules were visualized with a frame rate of 10 s−1. Data are representative of ≥ three independent experiments; scale bars, 5 µm. d, The foci resulting from TMR channel (mAzurite-biRhoBAST8) were analyzed with respect to their fluorescence intensity and diameter distribution, showing a single population of fluorescent species with diffraction-limited diameter (N = 3149 foci of 7 cells). The diffusion coefficients D were determined by analysis of TrackMate55 generated tracks (N = 922 tracks) with MSDanalyser56 in MatLab (MATHWORKS). Plotting the mean MSD vs. delay time and fitting a linear regression to the initial increase was used to calculate the diffusion coefficients with MSD = 4D·t56. e, The GFP foci (N = 7992 foci of 8 cells) and tracks (N = 997 tracks) resulting from the mAzurite-MS224:MCP-GFP signal were analyzed similar to d.
Extended Data Fig. 8 Formation of mRNA stress granules visualized by biRhoBAST–TMR2.
a, Live Cos7 cells expressing mAzurite-biRhoBAST8 mRNA were incubated with TMR2 (500 nM) and visualized by spinning disk confocal microscope (9.7 frames per second). After detection of single mRNA foci in a transfected cell, 500 µM sodium meta-arsenite was added and incubated for 65 min before the same cell was imaged again. Data are representative of two independent experiments; scale bars, 10 µm. b, The fluorescence distribution before treatment (upper graph) showed a single population of fluorescence intensity distribution (N = 268 foci), while the arsenite treated cells showed larger species with generally higher fluorescence (N = 143 puncta). c, The diameter analysis of untreated cells (upper graph) showed mostly diffraction limited spots (N = 268 foci) while the treated cells (N = 143 puncta, lower graph) showed mostly larger diameters. Analysis of the size of the individual foci was carried out using thresholded images with the Analyze Particles plugin in Fiji, following the plugin documentation53. The diameter d of the obtained areas A were calculated with d = 2·(A/π)0.5.
Extended Data Fig. 9 biRhoBAST–TMR2 enables single-molecule RNA visualization in fixed cells without changing the localization of the tagged mRNA.
a, Schematic illustration of the mAzurite mRNA tagged with biRhoBAST and the destination of the smFISH probes (green) hybridization sequences. b, Chemical structure of Atto647 that was used to label the smFISH probes. c, Confocal images of fixed Cos7 cells expressing mAzurite-biRhoBAST8 mRNA or mAzurite mRNA after incubation with smFISH probes (targeting the mAzurite sequence) and TMR2 (50 nM). Control experiments without adding smFISH probes were also performed in presence of TMR2 (50 nM). Data are representative of three independent experiments. d, Colocalization test of smFISH probes and TMR2 using JACoP54. A Pearson’s R value of 0.96 was obtained for the positive cell shown in the upper row in panel c that are also shown again for clarification. Scale bars, 5 µm.
Extended Data Fig. 10 biRhoBAST–TMR2 enables single-molecule detection of actin mRNA in Cos7 cells.
a, Schematic illustration of the mAzurite-β-actin mRNA tagged with biRhoBAST and the destination of the smFISH probes (green) hybridization sequences. b, Confocal images of fixed Cos7 cells expressing mAzurite-biRhoBAST8 mRNA or mAzurite mRNA from a CMV promoter after incubation with smFISH probes (targeting the mAzurite sequence) and TMR2 (50 nM). Control experiments without adding smFISH probes were also performed in presence of TMR2 (50 nM). Data are representative of two independent experiments. c, Colocalization test of smFISH probes and TMR2 using JACoP54. A Pearson’s R value of 0.90 was obtained for the positive cell shown in the upper row in panel b that are also shown again for clarification. d, Spinning disk confocal images of live Cos7 cells expressing low levels of mAzurite-β-actin-biRhoBAST8 or mAzurite-β-actin mRNA in the presence of 500 nM TMR2. Single mRNA molecules were visualized with a frame rate of 10 s−1. Data are representative of two independent experiments. e, The foci resulting from TMR channel (mAzurite-β-actin-biRhoBAST8) were analyzed with respect to their fluorescence intensity and diameter distribution, showing homogenous fluorescent species with a diffraction-limited diameter (N = 11855 foci of 15 cells). Scale bars, 5 µm.
Supplementary Video 1
Verification of single mRNA tagging with a biRhoBAST-MS2 tandem construct. Mobile puncta were imaged in live Cos7 cells co-expressing MCP-GFP and (i) mAzurite-biRhoBAST8-MS224 mRNA (ii) mAzurite-biRhoBAST8 mRNA (iii) mAzurite-MS224 mRNA were imaged in the presence of TMR2 (500 nM). Exposure time per frame: 100 ms. Scale bar, 5 µm.
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Bühler, B., Schokolowski, J., Benderoth, A. et al. Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging. Nat Chem Biol 19, 478–487 (2023). https://doi.org/10.1038/s41589-022-01228-8