Our finding that 2Me/PSLOOP MBs exhibit a marginal level of FPS raises the possibility of using MBs to image the dynamics and localization of single RNA transcripts in living cells with high accuracy. To determine whether 2Me/PSLOOP MBs can accurately detect single RNA transcripts, we developed a plasmid construct that encodes a transcript carrying an EGFP coding sequence followed by 32 tandem repeats of an MB target sequence (pEGFP-N1-32x) . As the MB target sequence and EGFP coding sequence are transcribed as one RNA molecule, we hypothesized that if MBs could hybridize to the engineered transcript, the target RNA should appear as a bright fluorescent spot reflecting MB-target hybridization. Furthermore, the MB fluorescent spot should colocalize with smFISH spots visualized using a set of probes targeting unique regions on the EGFP sequence. We found that following microporation of 2Me/PSLOOP MBs and smFISH processing, bright MB and smFISH spots could be detected in the nucleus and the cytoplasm (Figure 3A). Analysis of colocalization between MB and smFISH signals in three dimensions showed nearly 90% colocalization of the MB and smFISH signals, indicating that 2Me/PSLOOP MBs can detect engineered transcripts with high accuracy (Figure 3B). By contrast, in cells microporated with 2Me/PSFULL MBs and processed by smFISH, only 60% of the MB signals colocalized with smFISH signals. Thus, consistent with the analysis showing that 2Me/PSLOOP MBs generate lower FPS compared to 2Me/PSFULL MBs, 2Me/PSLOOP MBs can image single RNA transcripts more accurately than 2Me/PSFULL MBs in living cells.Figure 3Single-molecule RNA transcript detection using 2Me/PSLOOP MBs and smFISHHeLa cells stably expressing pEGFP-N1-32x were fixed and permeabilized 8 h or 24 h after microporation with 5 µM 2Me/PSLOOP MBs. smFISH was then performed with a pool of probes designed to target EGFP to assess MB detection accuracy for single RNA transcripts. A. Representative maximum intensity projection images of 2Me/PSLOOP MBs (Atto647NN-labeled) and EGFP smFISH probes (TAMRA-labeled) at 8 h or 24 h after microporation (Scale bar, 10 µm). B. The percentage of MB signals that colocalized with smFISH signals (left) and the percentage of smFISH signals that colocalized with MB signals (right) was analyzed on a cell-by-cell basis using a custom MATLAB program. Data are presented as mean ± SD from at least 10 cells. Significant difference from the 2Me/PSFULL MBs is indicated with asterisks (P < 0.05). The images are reproduced with permission from Elsevier .