Design of a stable Mango array

We have taken advantage of the recent isolation of brighter and more thermodynamically stable Mango aptamers28 to develop an RNA imaging array. We chose the Mango II aptamer (Fig. 1a) because of its resistance to formaldehyde fixation, enhanced thermal stability and high affinity for TO1-Biotin (TO1-B)28,29. As Mango II requires a closing stem to efficiently fold, we engineered modified stem sequences by altering the Watson–Crick base pairs of three adjacent aptamers to avoid potential misfolding between the aptamer modules (Fig. 1b, sequences in Supplementary Table 1). Bulk fluorescence intensity measurements of the resulting Mango II triplex array (M2x3) show a fluorescence intensity of ~2.5-fold relative to the Mango II monomer (Fig. 1c). The small intensity decrease likely resulting from homo-FRET. This was further tested by altering the linker length between two aptamers to modulate the relative orientation of each aptamer. In a Mango dimer, a single nucleotide linker is sufficient to fully recover the fluorescence (Supplementary Fig. 1). In a Mango triplex, however, the optimal spacer length for correct folding of the M2x3 construct was found empirically to be 5 nt, potentially due to reduced steric hindrance between monomers (Supplementary Fig. 1d). UV melting curves show identical folding stabilities of the M2x3 array relative to a single monomer (Fig. 1d), a marked improvement compared to a Broccoli triplex aptamer18 (Supplementary Fig. 1c-h). Similarly, the affinity for the TO1-B fluorophore is maintained in the M2x3 context as shown by measuring their apparent K D,app (Fig. 1e). Increasing the number of M2x3 repeats results in a proportional fluorescence intensity increase (Fig. 1c) showing that folding stabilities and fluorescence efficiencies are maintained in longer arrays. The M2x3 subunit was repeated eight times to directly compare fluorescent intensities with a 24-repeat MS2v5 construct14. With the in vitro confirmation of a thermodynamically and fluorescently stable tandem array, we set out to test the fluorescent stability of the tandem array in a cellular environment.

Fig. 1: In vitro characterisation of tandem Mango II arrays. a Crystal structure of Mango II aptamer (G-quadruplex—orange, Stem—grey) with TO1-Biotin bound (green). Taken from29 PDB ID: 6C63. b Diagram of Mango II and the Mango II x3 array with mutated bases purple and green. c Fluorescence increase of short Mango array constructs (40 nM) upon addition of an excess of TO1-Biotin (1 µM), error bars depict the standard deviation. d Differentials of the UV melting curves for each Mango array. e Titration of TO1-Biotin for each Mango construct, with a Hill equation fit to determine K d,app . Mango II—orange lines and black circles, Mango II x3—blue lines and black triangles. Full size image

Single-molecule imaging of Mango array-tagged mRNAs

To test the Mango array fluorescence in a cellular environment, we chose the mCherry coding sequence (CDS) as a reporter for mRNA transcription (Mango fluorescence) and translation (mCherry fluorescence). An M2x24 array was inserted downstream of the mCherry CDS/stop codon and the construct was placed under the expression of a doxycycline (dox) inducible promoter (Fig. 2a). Cos-7 cells were transfected with plasmid and induced with 1 µg/ml of dox for 1–4 h and either fixed and stained, or imaged in live cells in the presence of TO1-Biotin (200 nM). Upon induction, a significant increase in Mango specific fluorescence could be observed in both fixed and live cells (Fig. 2a). Individual diffraction-limited foci could be observed in both fixed and live cells, indicative of single mRNA molecules. Similarly, the observed foci shared a striking resemblance with the otherwise identical MS2v5x24 array in the presence of tdMCP-EGFP (Fig. 2b and Supplementary Fig. 2a, b). Furthermore, TO1-Biotin specific fluorescence was only apparent with expression of the mCherry-M2x24 mRNA and was absent with a control mCherry-MS2v5x24 mRNA (Fig. 2c).

Fig. 2: Cellular imaging of Mango II arrays. a Diagram of the mCherry-M2x24 construct + TO1-Biotin controlled by a doxycycline inducible promoter. Followed by representative fixed maximum projections (left & centre) and live cell images of observed cellular foci (right). b Diagram of the mCherry-MS2v5x24 construct + tdMCP-EGFP under the control of a doxycycline inducible promoter. Followed by representative fixed maximum projections (left & centre) and live cell images of observed cellular foci (right). c Diagram of the mCherry-MS2v5x24 construct + TO1-Biotin under the control of a doxycycline inducible promoter. Followed by representative fixed maximum projections (left & centre) and live cell images of observed cellular foci (right). For (a–c) TO1-Biotin signal is in yellow, EGFP signal is in green, mCherry is in red, Hoechst 33258 is in blue and scale bars = 10 µm and 1 µm inset. Fixed cell images depict expression in Cos-7 cells and live cell images depict expression in HEK 293 T cells. d Representative bleaching curve for an individual foci from a fixed Cos-7 cell, (yellow) fitted with a maximum likelihood step finding algorithm57 (black). e Distribution of 378 calculated bleaching step sizes from 70 individual mCherry-M2x24 foci from eight fixed cells. f Fixed cell maximum foci intensity distributions of mCherry-MS2v5x24 (black) and mCherry-M2x24 (yellow) in the presence of 200 nM TO1-Biotin. The M2x24 distribution quantifies~7000 foci from 13 cells. g Maximum live-cell foci intensities for cells expressing mCherry-M2x24 (yellow) and mCherry-MS2v5x24 (black) in the presence of 200 nM TO1-Biotin. n = 183 and 187 foci, respectively. Full size image

We determined the average monomer intensity based on step-photobleaching experiments of single foci in fixed cells as described previously28 (Fig. 2d), and obtained a value of ~40 a.u. in good agreement with our previously published value for the Mango II aptamer28 (Fig. 2e). The distribution of maximum foci intensities was acquired using FISH-quant30 using ~7000 foci from 13 cells. The distribution was distinct from the background observed in MS2v5x24 + TO1-B control cells (Fig. 2f). As expected from in vitro data28, the tdMCP-EGFP labelled foci are on average ~2-fold brighter than the TO1-Biotin dependent foci with both exhibiting a mean diffraction-limited diameter of ~250–300 nm (Supplementary Fig. 2c-e). The M2x24 intensity distribution can be fitted to the sum of three Poisson distributions, which estimates that on average, 7 ± 1 (36 ± 3%), 14 ± 1 (48 ± 3%) and 21 ± 1 (16 ± 3%) of the 24 aptamers in an array are active (Supplementary Fig. 2f). A similar distribution of intensities was observed in live cells, albeit with an ~1.5-fold increase in background fluorescence as shown with the MS2v5x24 + TO1-B control distribution and reduced fluorescence stability of the M2x24 arrays due to the increase in temperature (Fig. 2g, Supplementary Movie 1, 2). The nuclear and cytosolic speed distributions of the M2x24 and MS2v5-EGFPx24 foci in HEK293T cells were comparable (Supplementary Fig. 2g-h and Movie 3).

To further validate the observation of single molecules, we generated a M2/MS2-SLx24 RNA dual label construct encoding adjacent M2x24 and MS2-SLx24 arrays (Fig. 3a). Expression in Cos-7 cells was again induced using dox in the presence of transiently expressed NLS-tdMCP-mCherry. Cells were fixed and stained with 200 nM TO1-Biotin. Nuclear and cytoplasmic foci that were fluorescent in both green and red channels were observed; their size, intensity and localisation varied as expected for transcription sites, nuclear and cytoplasmic mRNAs (Fig. 3a). Removal of either the M2x24 or MS2-SLx24 arrays in either the presence or absence of tdMCP-mCherry confirmed the specificity of each of the fluorescent signals observed (Fig. 3b-d). As expected from the NLS, a bright saturating tdMCP-mCherry signal is observed in the nucleus for the majority of cells (Fig. 3b, d). Other cells showed far less background signal in the nucleus due to limited amounts of tdMCP-mCherry expression (Fig. 3a). This is in stark contrast to the uniform signal-to-noise ratio for both nuclear and cytosolic foci in cells expressing the Mango II array (Fig. 3c), highlighting the power of fluorogenic RNA systems for imaging nuclear RNAs.

Fig. 3: Validation of single-molecule imaging with M2/MS2 arrays. a Diagram and maximum projection of the M2/MS2-SLx24 array under the control of a dox inducible promoter and labelled with TO1-B (200 nM) and NLS-tdMCP-mCherry. Inset shows zoom of selected cytosolic area depicting colocalising signal. Scale bar = 10 µm and 2 µm inset. b Diagram and maximum projection of the MS2-SLx24 array in the presence of TO1-B (200 nM) and NLS-tdMCP-mCherry. c Diagram and maximum projection of the M2/MS2-SLx24 array in the presence of TO1-B (200 nM). d Diagram and maximum projection of the M2x24 array in the presence of TO1-B (200 nM) and NLS-tdMCP-mCherry. a–d All cells shown are PFA fixed Cos-7 cells, scale bars = 10 µm unless otherwise stated, TO1-B signal (green), mCherry signal (red) and Hoechst 33258 (blue). e Side by side comparison of TO1-B (green) and tdMCP-mCherry (red) labelled trajectories for an M2/MS2-SLx24 single molecule at 2 s and 23 s time points. Trace of the trajectory shown in blue and images taken from (Supplementary Movie 3). Scale bar = 4 µm. f XY axis plot of single molecule trajectory from (e) showing co-movement of fluorescent signal coloured as a function of time, TO1-B (green) and tdMCP-mCherry (red). g Plot of distance between foci localised in the TO1-B and mCherry channels (top) and speed (bottom) against time for the trajectory shown in (e) and (f). Average distance and standard deviation between foci plotted as shaded red line. h Fluorescence intensity distribution of M2/MS2-SLx24 foci from live cell tracking, TO1-B fluorescence (green) mCherry fluorescence (red) MS2-SLx24 + TO1-B background fluorescence (black). n = 17,544, 5881 and 33,370 foci, respectively, from 13 cells. i Signal-to-noise ratio distribution of foci detected in live cell M2/MS2-SLx24 tracking, TO1-B (green) and mCherry (red), n = 17,544 and 5881 respectively from 13 cells. Full size image

Live-cell imaging of the M2/MS2-SLx24 RNA in the presence of both TO1-B and tdMCP-mCherry showed multiple coincident foci per cell (Supplementary Fig. 3a and Supplementary Movie 4), which were fluorescent in both green and red channels. Foci could be readily tracked using basic widefield microscopy and the TrackMate ImageJ plugin31, with trajectories analysed at 226 ms per frame. Individual trajectories show excellent colocalisation of the foci over long spatial trajectories and times (Fig. 3e, f and Supplementary Movie 5). Plotting the difference between the xy position of the Mango and MS2 foci depicts an average difference of ~250 nm with larger fluctuations above the standard deviation only at the highest diffusional speeds and likely resulting from the sequential frame acquisition of the microscope (Fig. 3g). Analysis of mean squared displacement (MSD) values for ~1000 trajectories from multiple cells expressing M2/MS2-SLx24 and labelled with tdMCP-mCherry show a broad distribution of diffusive speeds (Supplementary Fig. 3b). The increased signal-to-noise in the Mango channel further enhanced the quality of foci detection and length of subsequent tracking (Supplementary Fig. 3c, d and Supplementary Movie 6). Due to the NLS, a strong tdMCP-mCherry signal in the nucleus complicates the analysis of single-molecule trajectories in both the nucleus and cytosol, which requires adjusting the TrackMate plugin31 thresholds on a cell-by-cell basis, as described in materials and methods. The nuclear foci observed above the background in the mCherry channel (blue distribution) have a slow diffusive behaviour with a mean MSD = 0.062 ± 0.019 µm2/s and a mean intensity ~6-fold greater than that expected for a single mRNA molecule suggesting that they correspond to transcription sites (Supplementary Fig. 3e and Supplementary Movie 7). In contrast the cytosolic foci detected in the mCherry channel have a mean MSD = 0.464 ± 0.029 µm2/s and an intensity distribution with a single peak, both indicative of freely diffusing single molecules. M2/MS2-SLx24 foci detected across the entire cell using TO1-B fluorescence (yellow distribution), show a broader distribution of MSD sharing similarities of both nuclear and cytosolic distributions described previously with a mean MSD = 0.122 ± 0.077 µm2/s. Further confirmation of slow diffusing molecules was observed with data acquired at a 3.6 s time frame rate (black distribution) which have a mean MSD = 0.089 ± 0.010 µm2/s. As expected, the difference in MSD between M2/MS2-SLx24 and M2x24 arrays imaged in the presence of TO1-B was negligible (Supplementary Fig. 3b, f and Supplementary Movie 4). Quantification of intensities for both M2/MS2-SLx24 foci in live cells shows that both TO1-B and mCherry distributions are distinct from an MS2-SLx24 array in the presence of TO1-B (Supplementary Fig. 3g). The M2/MS2-SLx24 + TO1-B shows a marginally brighter distribution in the Mango channel than the mCherry channel as expected due to mCherry’s ~2-fold lower brightness than EGFP and its reduced photostability32 (Fig. 3h). Quantification of the signal-to-noise ratio of each M2/MS2-SLx24 transcript detected shows a marked increase in the M2x24 + TO1-B channel over the MS2-SLx24 + tdMCP-mCherry channel (Fig. 3i). Taken together, these data show that M2x24 arrays enable the detection and tracking of single mRNA transcripts in live cells and clearly illustrate the benefits in using fluorogenic RNA imaging strategies.

Mango arrays do not affect localisation of β-actin mRNA

To test the ability of Mango arrays to recapitulate the localisation pattern of biological mRNAs, we inserted an M2x24 array downstream of the 3′UTR of an N-terminally mAzurite labelled β-actin gene (Fig. 4a). The β-actin 3′UTR contains a zipcode sequence that preferentially localises the mRNA at the edge of the cell or the tips of lamellipodia33,34,35,36. In addition, we tagged the β-actin coding sequence with a N-terminal Halotag to validate the translation of the β-actin mRNA in fixed cells. Upon transient expression of both tagged β-actin-3’UTR-M2x24 constructs in Cos-7 fibroblast cells, a specific increase in Mango fluorescence could be observed when compared to a equivalent construct containing an MS2v5x24 cassette in the presence of TO1-B (Supplementary Fig. 4a-c). Incubation with the HaloTag-TMR (Tetramethylrhodamine) ligand gave rise to cells which were efficiently and specifically labelled with TMR. The TMR signal could be seen to accumulate at the periphery of the cells and form cytosolic filaments in both M2x24 and MS2v5x24 labelled mRNAs, confirming the faithful translation and targeting of the Halo-β-actin mRNAs. As expected, the Mango specific signal of the β-actin-3’UTR-M2x24 mRNAs preferentially localise at the edge of the cell or the tips of lamellipodia (Fig. 4b and Supplementary Fig. 4a-c), showing that the array did not affect mRNA transport in either of the constructs. To quantify the mRNA localisation, we calculated the polarisation index (PI) for each localised mRNA, as adapted from a recently reported method37 (see methods). Both MS2v5-EGFP and M2x24 labelled β-actin mRNAs show a significantly higher PI than β-actin mRNAs lacking the 3’UTR and the mCherry-M2x24 construct negative control (Fig. 4b, c), in good agreement with previous results36 indicating that the Mango array does not interfere with RNA localisation.

Fig. 4: Imaging polarisation of β-actin mRNAs in fibroblasts. a Diagram of the mAzurite labelled β-actin- ± 3′UTR-M2x24 construct under the control of a constitutive promoter. b Representative fixed cell images of the localisation patterns for the mAzurite-β-actin-3’UTR-MS2v5x24—EGFP (top left), mAzurite-β-actin-3′UTR-M2x24 (top right) mAzurite-β-actin-∆3′UTR-M2x24 (bottom left) and mCherry-M2x24 (bottom right) constructs in Cos-7 cells. Stained with TO1-B (200 nM—green channel), TMR or AlexaFluor® 647-Phalloidin (165 nM—red channel) and Hoechst 33258 (1 µg/ml – blue channel), scale bars = 10 µm. Mean PI calculation for RNA localisation within the depicted cell shown in white inset. c Mean PI for each Mango tagged construct for multiple cells (n indicated), error bars depict the standard error in the mean and p-values calculated using a two-tailed student’s T-test. d Diagram of Quasar® 670 (Q670 - magenta) and Alexa Fluor™ 488 (AF488—green) smFISH probes against the TMR labelled Halo-β-actin-3’UTR-M2x24 construct under the control of a doxycycline inducible promoter. e Maximum projection of a cell expressing Halo-β-actin-3’UTR-M2x24 mRNA and visualised with β-act-Q670 (magenta) and M2-AF488 (green) smFISH probes. Scale bars = 10 µm and 2 µm for high magnification image (lower right panel). f Total number of RNA foci in dually labelled Halo-β-actin-3′UTR-M2x24 with smFISH AF488 and Q670 probes using FISH-quant. Single cells foci numbers depicted as grey data points, a line with a gradient = 1 (solid red) and data points fit with a straight line (dashed black—slope = 0.88, r2 = 0.7). g Calculated fraction of overlapping signals from multiple dually labelled mRNAs with n as number of cells, error bars depict the standard deviation. RNA construct labelled in black with the corresponding smFISH probes in magenta (Q670) and green (AF488). Full size image

To validate the presence of full-length β-actin transcripts the Mango arrays were imaged in conjunction with Stellaris® single-molecule RNA FISH probes against β-actin CDS, mCherry CDS, M2x24 array, or MS2v5x24 array38,39 (Fig. 4d). Use of these probes validated the presence and accurate detection of full-length β-actin transcripts with either the MS2v5x24 or the M2x24 arrays (Fig. 4e and Supplementary Fig. 4d–f). We quantified the total number of foci detected in Alexa FluorTM 488 (AF488) and Quazar® 670 (Q670) channels for probes against the M2 array and mCherry CDS respectively, in cells expressing mCherry-M2x24 (n cell = 27). The data shows a positive correlation close to a slope of 1 (0.88) and an r2 = 0.7, indicating that the majority of foci correspond to full-length transcripts (Fig. 4f). Similarly, the percentage of transcripts with overlapping signals in cells expressing β-act-3’UTR-MS2v5x24, β-act-3’UTR-M2x24 and mCherry-M2x24 RNAs was high at ~70% (Fig. 4g). As a control, the β-actin-Q670 probes detecting endogenous β-actin mRNA were used in conjunction with M2-AF488 probes in cells expressing the mCherry-M2x24 construct. As expected, this control exhibited a significant decrease in the amount of overlapping signal to ~10% (Fig. 4g and Supplementary Fig. 4g). Taken together these experiments show that the tandem Mango arrays do not affect the cellular localisation of β-actin mRNA and that the tag is efficiently retained within the transcript to a level similar to that of established MS2v5 cassettes.

Super-resolution RNA imaging with Mango arrays

Due to the exchange of TO1-B and subsequent fluorescent recovery in fixed cell samples, extended imaging times under pulsed illumination can be achieved28. We took advantage of this fluorescence recovery and used structural illumination microscopy (SIM) to acquire Mango tagged RNA images at super-resolution (~100 nm)40,41. To first test the recovery of Mango array fluorescence, the mCherry-M2x24 construct was imaged with constant wave illumination for 5 s followed by a 5 s pause to allow for fluorophore recovery. Images were acquired in either PKM buffer (10 mM sodium phosphate, 140 mM KCl, 1 mM MgCl 2 ), Vectashield liquid mounting media or ProLong Diamond hard setting mounting media (Fig. 5a). As expected, we saw efficient recovery of Mango fluorescence in PKM buffer as described previously28. Conversely, the hard setting ProLong media showed no fluorescence recovery consistent with fluorophore exchange. Similarly, the Vectashield liquid media reduced fluorescence recovery to an extent that would ultimately lead to the complete loss of Mango signal over the course of a structural illumination acquisition. Therefore, PKM buffer was used in further SIM experiments.

Fig. 5: Super resolved imaging of Mango tagged RNAs. a Bleaching curves of individual mCherry-M2x24 foci in fixed HEK293T cells using different media (PKM buffer—yellow, Vectashield® - blue and Prolong® Diamond – black) upon 5 s intervals of constant illumination. b Widefield and SIM maximum projections of the β-actin-∆3′UTR-M2x24 construct (green) costained with Phalloidin-AF647 (red) and Hoechst 33258 (blue), scale bar = 10 µm. HEK293T cells. c Mean intensity bleaching curves of Hoechst, Mango and Phalloidin signal during structured illumination acquisition of β-actin-∆3’UTR-M2x24 expressing HEK293T cells. The standard deviation is plotted as the shaded area around each curve obtained from eight different fields of view. d Widefield and SIM maximum projections of NEAT-1 v1-M2x24 construct (green) costained NONO (magenta) and Hoechst 33258 (blue) in HEK293T cells. Zoom of highlighted foci shown as inset. Large scale bars = 5 µm, inset scale bars = 0.5 µm. Inset intensity profile (lower right) of annotated NONO foci (magenta) from SIM maximum projection showing the peripheral NEAT-1 signal (green). Full size image

During SIM acquisition, Mango tagged β-actin mRNA was imaged in conjunction with Hoechst 33258 and Phalloidin-AF647 to stain the nucleus and actin cytoskeleton respectively (Fig. 5b). The resulting images reveal well-defined foci below the diffraction limit (~100 nm). SIM reconstruction did not to affect the observed PI as confirmed by using a side by side comparison of widefield and SIM images (Supplementary Fig. 5a-c). Using the SIMcheck ImageJ plugin42, the total loss of fluorescence during acquisition was measured for multiple cells (Fig. 5c). A mean fluorescence loss of ~20% was seen in the Mango channel over the entirety of image acquisition. The photostable Hoechst dye showed minimal loss at ~5%, whereas the Phalloidin-AF647 signal dropped ~55% over the imaging period. This demonstrates the feasibility of using the stable exchange of RNA Mango TO1-B fluorophores to efficiently reconstruct SIM images—an exciting advance as stable exchange with protein based fluorophore aptamers have not succeeded to date owing to significant photodamage of the protein aptamer tag43.

Due to the high resolution and enhanced fluorescent contrast obtained within the nucleus, we aimed to image a nuclear retained lncRNA with SIM. To this end, the short isoform of the NEAT-1 lncRNA (NEAT-1v1 - 3.7 kb) was chosen and labelled with the M2x24 array. NEAT-1 is an interesting candidate due to its well-known nuclear coordination of paraspeckles, which has been well characterised using SIM44. Upon mild induction, multiple small foci could be observed in the nucleus indicative of nascent transcripts and active transcription. Upon stronger induction, the formation of large Mango specific nuclear foci was observed. Immunostaining for the paraspeckle associated protein NONO (Non-POU domain-containing octamer-binding protein) showed a diffuse colocalization of both signals (Fig. 5d). Upon SIM reconstruction, the NEAT-1-v1 RNA could be seen to surround the core of the paraspeckle containing the NONO protein. Similarly, small microspeckles could be observed outside of the larger paraspeckles. Quantification of the number of NONO foci with adjacent NEAT-1-v1-M2x24 signal showed ~70% of foci had associated lncRNA signal (Supplementary Fig. 5d, e). Given the use of plasmid expression for NEAT-1-v1-M2x24, a full association with paraspeckles was not to be expected. Together, these images are in good agreement with previously published RNA FISH images of the short isoform of NEAT-1 associating with the paraspeckle outer shell and formation of microspeckles45. These data demonstrate that the fluorescence recovery of the Mango aptamers can be used to enhance the reconstruction of super-resolved images. Additionally, the improvement in resolution is not solely contained to the use of SIM, but also that the fluorogenic properties of Mango enable higher contrast imaging of RNAs as previously speculated46 especially within the nucleus where nuclear localisation of FP-MS2 can preclude RNA detection at the single-molecule level.