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RNA-stabilized coat proteins for sensitive and simultaneous imaging of distinct single mRNAs in live cells

Nature Methods (2025)Cite this article

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Abstract

RNA localization and regulation are critical for cellular function, yet many live RNA imaging tools suffer from limited sensitivity due to background emissions from unbound probes. Here we introduce conditionally stable variants of MS2 and PP7 coat proteins (which we name dMCP and dPCP) designed to decrease background in live-cell RNA imaging. Using a protein engineering approach that combines circular permutation and degron masking, we generated dMCP and dPCP variants that rapidly degrade except when bound to cognate RNA ligands. These enhancements enabled the sensitive visualization of single mRNA molecules undergoing differential regulation within various subcompartments of live cells. We further demonstrate dual-color imaging with orthogonal MS2 and PP7 motifs, allowing simultaneous low-background visualization of distinct RNA species within the same cell. Overall, this work provides versatile, low-background probes for RNA imaging, which should have broad utility in the imaging and biotechnological utilization of MS2-containing and PP7-containing RNAs.

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Fig. 1: Design of an RNA-dependent conditionally stable MS2 coat protein.
Fig. 2: Single mRNA detection of MS2-tagged transcripts in cells co-expressing 4×mNG–dMCP.
Fig. 3: dMCP-based imaging NRE-containing mRNA transcripts.
Fig. 4: Live-cell tracking of MS2-tagged single mRNAs in 4×mNG–dMCP-expressing U2OS cells.
Fig. 5: Design of dPCP and two-color RNA imaging using dMCP and dPCP.

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Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Plasmid DNA and detailed sequence information for selected constructs are available through Addgene. Source data are provided with this paper.

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Acknowledgements

This work was funded by the National Institutes of Health (NIH) through National Institute of General Medical Sciences research grant R35GM128859 (to J.T.N.). C.J.K. was supported through the Boston University training program in Quantitative Biology and Physiology (NIH grant T32GM008764) and a fellowship from the Multicellular Design Program (Boston University Kilachand Fund). A.M.M. was the recipient of a National Science Foundation Graduate Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Christopher J. Kuffner, Alexander M. Marzilli.

Authors and Affiliations

  1. Department of Biomedical Engineering, Biological Design Center and Center for Multiscale and Translational Mechanobiology, Boston University, Boston, MA, USA

    Christopher J. Kuffner, Alexander M. Marzilli & John T. Ngo

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  1. Christopher J. Kuffner
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  2. Alexander M. Marzilli
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Contributions

All authors contributed to the design of experiments, analysis of the results and preparation and editing of the manuscript. C.J.K. and A.M.M. executed experiments. J.T.N. supervised the work.

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Correspondence to John T. Ngo.

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The authors are co-inventors on a patent application filed by the Trustees of Boston University (US patent application no. 18/813,643).

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Nature Methods thanks Gal Haimovich and the other, anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 MS2-binding analysis using NLS-cpMCP-S52.

(a) Schematic depicting the RNA binding-mediated nuclear export of NLS-cpMCP-S52, which is imported into and retained within the nucleus in its unbound form. Upon overexpression and nuclear export of MS2-tagged mRNA, protein-RNA binding leads to translocation of NLS-cpMCP-S52 to the cytoplasm. (b) Widefield fluorescence microscopy images of HEK293FT cells co-transfected to express mVenus-NLS-cpMCP-S52 and either H2B-mCherry-pA or H2B-mCherry-16xMS2-pA transcripts show that expression of MS2-tagged mRNA leads to nuclear exclusion of NLS-cpMCP-S52, indicating that it is capable of binding to MS2 loops. Insets in the bottom right display H2B-mCherry expression (c) HEK293FT cells were transfected to express mVenus-cpMCP-S52 (with no NLS) or the degron-appended mVenus-dMCP-S52 (both lacking an NLS). These cells were cotransfected to express H2B-mCherry-pA or H2B-mCherry-16xMS2-pA transcripts. The images show that the fluorescence of mVenus-dMCP-S52 is heavily reduced in the absence of MS2 RNA, and that the fluorescence is partially recovered in the presence of MS2 RNA.

Extended Data Fig. 2 Extended data of dMCP with various fluorescent and bioluminescent fusion partners.

(a) Full-well views of the HEK293FT cells shown in Fig. 1f display dMCP’s sustained performance across a large population of transfected cells. Tiled fluorescence microscopy images of five dMCP fusion proteins are shown, with fluorescence emissions ranging from cyan to far-red wavelengths. The signal from mCherry or mTurq2 cotransfection markers are shown in the bottom right windows. (b) MS2-RNA stabilization of an NLuc-dMCP fusion construct. Relative NLuc fluorescence was normalized to Fluc co-transfection marker and quantified by plate reader. HEK293FT cells were co-transfected with NLuc-MCP variants and either H2B-mCherry-pA (gray circles) or H2B-mCherry-16xMS2-pA (red circles). Individual points, bars, and error bars represent the measured intensity, mean and S.D. of n = 3 independent transfections.

Source data

Extended Data Fig. 3 Extended data comparing dMCP and HCR single-molecule RNA puncta.

(a-c) Maximum intensity projection confocal microscopy images of fixed U2OS cells expressing H2B-mCherry-24xMS2 RNA tagged by 4xmNG-HA-dMCP and HCR probes targeting mCherry. dMCP signal is shown on the leftmost column (green), HCR signal shown left-of-center (magenta), merged images are shown right-of-center, and an overlay with digitally-labeled cytosolic spots as determined by ImageJ’s particle analysis plug-in is shown on the rightmost column. In the overlayed images, green spots are dMCP spots that do not overlap HCR, magenta spots are HCR spots that do not overlap dMCP, and white spots are spots from both channels which overlap. H2B-mCherry signal is shown in the bottom right. (d) Signal-to-noise ratio (SNR) of dMCP spots compared with HCR spots across three cells. SNR is measured as the average intensity of a spot divided by the average intensity of the entire cytosolic region excluding spots. Both values are calculated with the average background intensity of the local culture medium, calculated as the MFI of a large boxed region in each image in which no cells were detected, subtracted. Bars represent the mean of each population. (c) Area in square micrometers of dMCP spots in comparison to HCR spots. Bars represent the mean of each population. (d) Tabulated values for co-localization between dMCP and HCR spots. dMCP spot counts per cell are a; n = 182, b: n = 299, c: n = 447. HCR spot counts per cell are a: n = 138, b: n = 269, c: n = 345. The lower HCR spot count is primarily due to closely spaced, large puncta that were collectively counted as single foci because they could not be individually resolved.

Source data

Extended Data Fig. 4 Confocal images and spot analysis comparing 1xmNG-dMCP to 1xmNG-NLS-tdMCP.

(a) Maximum intensity projection confocal microscopy images of fixed U2OS cells expressing H2B-mCherry-24xMS2-pA RNA tagged by 1xmNG-HA-dMCP (top two rows, images D1-D10) or 1xmNG-HA-NLS-tdMCP (bottom two rows, images N1-N10). Images are displayed with signal inverted to better preserve visibility of spots after image compression below original 2046-pixel width. (b) Zoomed images displaying regions outlined by boxes in (a). These images more accurately display the intensity and size of spots as they appear in the original images. (c,d) Signal-to-Noise Ratio (SNR) of spots in all displayed (c) dMCP and (d) NLS-MCP cells. SNR is measured as the Mean Fluorescent Intensity (MFI) of pixels within a spot divided by the mean fluorescent intensity of the entire cytosolic region excluding spots and the nucleus (see Supplementary Fig. 6 for a portrayal of how this region is defined). Both values are calculated with the average background intensity of the local culture medium (calculated as the MFI of a large boxed region in each image in which no cells were detected) subtracted. Bars represent the mean of each population. (e,f) Area in square micrometers of (e) dMCP and (f) NLS-MCP spots. Bars represent the mean of each population. (g,h) MFI of (e) dMCP and (f) NLS-MCP spots. Bars represent the mean of each population. (i) Comparison of mean SNR between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: *** P = 0.0002. (j) Comparison of mean spot area between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ns P = 0.1073. (k) Comparison of mean spot MFI between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ns P = 0.7412. (l) Comparison of MFI for cytosol (as described above) between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ****P = 8.9 × 10−5. (m) Comparison of noise- calculated cytosol MFI minus average background intensities of local culture medium (as described above) between the ten dMCP and ten NLS-MCP images. Statistical significance determined by unpaired two-tailed Student’s t-test: ** P = 0.0010. Spot counts for each image are D1: n = 111, D2: n = 165, D3: n = 284, D4: n = 444, D5: n = 192, D6: n = 70, D7: n = 70, D8: n = 69, D9: n = 115, D10: n = 177, N1: n = 132, N2: n = 308, N3: n = 320, N4: n = 88, N5: n = 116, N6: n = 191, N7: n = 209, N8: n = 109, N9: n = 86, N10: n = 150.

Source data

Extended Data Fig. 5 Expanded images of mRNAs exhibiting varying degrees of nuclear retention as revealed by dMCP.

Here, expanded views of the samples in Fig. 3a. are shown in order to display a larger population of cells with dMCP-labeled mRNA. HEK293FT were transfected to express mNG-HA-dMCP and mRNAs that show differing levels of nuclear retention depending on the presence of a MEG3 Nuclear Retention Element and/or introns.

Extended Data Fig. 6 Nuclear speckle co-localization analysis of MEG3NRE-β-Globin-24xMS2-pA mRNA tagged with mNG-HA-dMCP, mNG-HA-tdMCP, or mNG-HA-NLS-tdMCP.

Widefield fluorescence microscopy images of fixed U2OS cells transfected to express MEG3NRE-β-Globin-24xMS2-pA mRNA and (a) 1xmNG-HA-dMCP, (b) 1xmNG-HA-tdMCP, or (c) 1xmNG-HA-NLS-tdMCP. Nuclear speckles were stained using anti-SC35 primary antibody and an AlexaFluor-647 (AF647)-conjugated rabbit anti-mouse secondary antibody. Signals from mNG-tagged MCP variants are displayed in green, and signals from AF-647 stained nuclear speckles are displayed in magenta. The intensity profiles below each image are measured for the regions indicated by yellow arrows, with values normalized to the maximum intensity of each individual trace.

Source data

Extended Data Fig. 7 Widefield live single-molecule RNA imaging comparison of MCP variants.

Widefield fluorescence microscopy images of live U2OS cells transfected to express mCherry-24xMS2-pA mRNA and HA-dMCP (left), HA-NLS-tdMCP (center), or HA-tdMCP (right) fused to (a) 1xmNG or (b) 4xmNG. All images are single frames extracted from live recordings that were obtained using the same microscope settings throughout. Images are displayed with multiple brightness settings to more fully portray the relative image quality produced by each variant.

Extended Data Fig. 8 Stabilization of dMCP by RNA containing reduced-affinity MS2 loops.

(a) Generalized ribonucleotide sequences for high affinity C-variant and reduced affinity U-variant MS2 loops. ‘S’ corresponds to G or C, ‘H’ corresponds to A,U, or C, and ‘N’ corresponds to any nucleotide. (b) Representative flow cytometry data of the dMCP stabilization response to C-variant MS2 RNA (BFP-24xMS2(C)-pA, red), U-variant MS2 RNA (BFP-24xMS2(U)-pA, blue), or control BFP RNA (BFP-pA, gray). mScarlet-dMCP intensity is plotted over BFP intensity. (c) Median mNG-normalized mScarlet-dMCP intensities for the BFP positive population of cells in the experiment described in (e). Quantification performed by flow cytometry in HEK293FT cells co-transfected to express BFP mRNA containing C-variant MS2 stem loops (red circles), U-variant MS2 stem loops (blue circles) or no MS2 stem loops (gray circles) in combination with the dMCP reporter construct. Each point represents the median mNG-normalized mScarlet expression for independent transfections. Bars and error bars represent the mean and S.D of three independent transfections (n = 3). (d) Widefield fluorescence microscopy images of live U2OS cells transfected to express BFP-24xMS2(C)-pA or BFP-24xMS2(U)-pA with 4xmNG-dMCP. Images are single frames extracted from live recordings that were obtained using the same microscope settings.

Source data

Extended Data Fig. 9 Subcellular distribution of LSS-mCherry-KDEL-16xMS2 mRNA movements relative to distribution of LSS-mCherry-KDEL protein expression.

Data from three different regions of live U2OS cells expressing 4xmNG-dMCP and LSS-mCherry-KDEL-16xMS2-pA RNA. 4xmNG-dMCP signal is displayed in the top row and LSS-mCherry-KDEL signal is displayed in the middle row. The bottom row depicts RNA particle traces color-coded by apparent diffusion coefficient as previously described and overlaid on top of LSS-mCherry-KDEL signal. It can be seen that most dMCP-labeled RNA particles in these cells are static, and that they tend to accumulate in regions with higher LSS-mCherry-KDEL signal.

Source data

Extended Data Fig. 10 Subcellular distribution of TOM20-mCherry-16xMS2 mRNA movements relative to distribution of TOM20-mCherry protein expression.

Data from three different regions of live U2OS cells expressing 4xmNG-dMCP and TOM20-mCherry-16xMS2-pA RNA. 4xmNG-dMCP signal is displayed in the top row and TOM20-mCherry signal is displayed in the middle row. The bottom row depicts RNA particle traces color-coded by apparent diffusion coefficient as previously described and overlaid on top of TOM20-mCherry signal. It can be seen that dMCP-labeled RNA particles in these cells are in visibly distinct static and moving populations, with static RNA tending to concentrate near regions of high TOM20-mCherry signal and moving RNA tending to be found in regions lacking this signal.

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Supplementary Figs. 1–16 and amino acid sequences for the reported protein domains

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Supplementary Video 1

U2OS cells transfected to express dMCP, NLS–tdMCP or tdMCP, each fused to mNG or a 4×mNG tandem repeat. All cells also express H2B–mCherry–24×MS2–pA RNA. The videos are replayed with multiple brightness settings to display the relative image quality produced by the MCP variants and fusions. 50-ms exposure time, 2 × 2 binning.

Supplementary Video 2

U2OS cells transfected to express H2B–mCherry–24×MS2–pA mRNA and dMCP fused to 1×mNG, a 2×mNG tandem repeat or a 4×mNG tandem repeat. Videos of cells were recorded using three different settings (50-ms exposure/1 × 1 binning, 50-ms exposure/2 × 2 binning and 200-ms exposure/1 × 1 binning). All videos are replayed through four different brightness settings in series to best portray the relative image quality produced by the various dMCP fusions under different microscopy settings.

Supplementary Video 3

U2OS cells transfected to express 4×mNG–dMCP and H2B–mCherry–24×MS2–pA RNA, in which the MS2 loops are either the high-affinity C-variant or the reduced-affinity U-variant. 50-ms exposure time, 1 × 1 binning.

Supplementary Video 4

HDF-neo primary cells transduced to express 2×mNG–dMCP and CFP–24×MS2–pA mRNA. 100-ms exposure time, 1 × 1 binning.

Supplementary Video 5

U2OS cells transfected to express mVenus–dMCP and H2B-mCherry–24×MS2–pA mRNA. Cells were also transfected with plasmid encoding VHHGFP4–CAAX, a nanobody that binds with mVenus fused to a C-terminal prenylation motif for membrane anchoring or control DNA. 200-ms exposure time, 2 × 2 binning; playback speed is 4.4× real time.

Supplementary Video 6

U2OS cells transfected to express 4×mNG–dMCP and H2B–mCherry–16×MS2–pA. Cells were imaged before and after 30-minute treatment with the translational repressor harringtonine. 50-ms exposure time, 1 × 1 binning.

Supplementary Video 7

U2OS cell transfected to express 4×mNG–dMCP and LSS–mCherry–KDEL–16×MS2–pA. In this cell, dMCP-tagged LSS–mCherry–KDEL–16×MS2–pA mRNA appears largely anchored to regions of high LSS–mCherry–KDEL expression. 50-ms exposure used time for 4×mNG–dMCP; 200-ms exposure time used for TOM20–mCherry. 1 × 1 binning.

Supplementary Video 8

U2OS cell transfected to express 4×mNG–dMCP and LSS–mCherry–KDEL–16×MS2–pA. Cells were imaged before and after 30-minute treatment with the translational repressor harringtonine. 50-ms exposure time, 1 × 1 binning.

Supplementary Video 9

U2OS cell transfected to express 4×mNG–dMCP and TOM20–mCherry–16×MS2–pA. In this cell, dMCP-tagged TOM20–mCherry–16×MS2–pA mRNA moves more slowly in subcellular regions of high TOM20–mCherry expression. 50-ms exposure time used for 4×mNG–dMCP; 200-ms exposure time used for TOM20–mCherry. 1 × 1 binning.

Supplementary Video 10

U2OS cell transfected to express 4×mNG–dMCP and TOM20–mCherry–16×MS2–pA. Cells were imaged before and after 30-minute treatment with the translational repressor harringtonine. 50-ms exposure time, 1 × 1 binning.

Supplementary Video 11

HEK293FT cell with CRISPR knock-in of TOM20–mCherry–10×MS2 at the endogenous TOM20 locus. Arrows indicate regions of TOM20–mCherry signal co-localizing with dMCP-labeled TOM20 transcripts over several seconds. The two videos were recorded simultaneously, with 50-ms exposure time used for imaging 4×mNG–dMCP and 100-ms exposure time used for imaging TOM20–mCherry. 1 × 1 binning.

Supplementary Video 12

U2OS cells transfected to express dPCP, NLS–tdPCP or tdPCP, each fused to mNG or a 4×mNG tandem repeat. All cells also express H2B–mCherry–18×PP7–pA RNA. The videos are replayed with multiple brightness settings to display the relative image quality produced by the PCP variants and fusions. 50-ms exposure time, 2 × 2 binning.

Supplementary Video 13

U2OS cell transfected to express 2×Halo–dMCP, 4×mNG–dPCP, H2B-mCherry–24×MS2–pA and LSS–mTurq2–18×PP7–pA. 200-ms exposure time and 2 × 2 binning were used for both channels. Videos were recorded back to back to avoid loss of framerate that occurs due to switching of filter cubes.

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Kuffner, C.J., Marzilli, A.M. & Ngo, J.T. RNA-stabilized coat proteins for sensitive and simultaneous imaging of distinct single mRNAs in live cells. Nat Methods (2025). https://doi.org/10.1038/s41592-025-02782-4

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