Calibration experiments to relate TFI measurements to single molecule and single transcript numbers were carried out as follows: HEK293 cells were transfected with pEGFP-C1 (Clontech) and harvested on ice the next day. Cytoplasmic extract was prepared by swelling and lysing cells in RSB(resuspension buffer)buffer (10 mM tris, 10 mM NaCl, and 3 mM MgCl2). Cytoplasmic extract was then diluted in 1× phosphate-buffered saline (PBS) at 1:50 to 1:1000, and 1 to 2 μl were placed between two 30-mm no. 1.5 coverslips (Thermo Fisher Scientific) and placed in a PeCon POCmini-2 cell chamber as described (34). The chamber was placed in the same stage incubator as described above, and GFP molecules were imaged with the same laser intensity as in the live cell experiments but with exposure times of 1000 ms over 100 time points in one plane. Image series were analyzed using the StaQTool to detect fluorescent objects and measure TFI values. Single GFP molecules were identified by detecting events irreversible and rapid loss of fluorescence bleaching, where the corresponding difference of the TFI value was fitted and recorded as TFI of a single GFP molecule (fig. S4A). Live cell experiments to determine the labeling rate of single reporter gene transcripts were performed using PROP and EX2 reporter cell lines transiently transfected with MS2-GFP or PP7-GFP, respectively. One day after transfection, the reporter gene transcription was induced with doxycycline (0.5 μg/ml) for 1 hour, and single imaging plane time series of 100 time points with 500-ms intervals were recorded. Particles with high mobility in the cell nucleus represent single-labeled mRNA transcripts in contrast to stationary larger objects representing sites of reporter gene transcription with multiple-labeled RNAs present. Analysis of the brightness of single-labeled reporter gene mRNAs was performed using the STaQTool by determining the TFI of single-labeled mRNA molecules in the image frame (fig. S4B). The mean TFI value of single GFP molecules was related to the mean TFI of MS2-GFP– or PP7-GFP–labeled transcripts to determine the labeling ratio (fig. S4C). Additional live and fixed cell calibration measurements to detect single mRNA transcripts labeled by MS2-GFP, MS2-RFP, and PP7-GFP were performed accordingly to determine the single transcript labeling mean TFI values for the respective labeling protein and imaging settings used in the live cell I–SceI cutting assays (fig. S5). Particles with high mobility in the cell nucleus represent single-labeled mRNA transcripts in contrast to stationary larger objects representing sites of reporter gene transcription with multiple-labeled RNAs present (fig. S5, A to D). To improve the TFI measurements of double-labeled transcripts from the EX2 reporter gene, cells were double transfected (MS2-RFP and PP7-GFP) and, after transcription induction for 1 hour, fixed for 10 min in 3.7% formaldehyde, washed in PBS, and embedded in VECTASHIELD mounting medium (Vector Laboratories). Image stacks of 30 to 40 optical slices at 0.27-μm intervals were taken using the same imaging conditions as in live cell experiments. Analysis was performed using the STaQTool by determining the TFI of single-labeled mRNA molecules in the image frame (live cells) or plane (fixed cells) of highest intensity (fig. S5, E and F) in cells, where the site of reporter gene transcription was identified as a much larger and brighter spot within the nucleus and excluded from analysis. Our results show that around 13 (MS2) to 15 (PP7) of 24 RNA stem loops were occupied, on average, and confirm a less than 100% labeling ratio detected before.

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