Given their ability to detect single engineered RNA transcripts with high accuracy in live cells, 2Me/PSLOOP MBs may be a promising tool to study the trafficking and localization of single RNA transcripts in real time. Figure 4A shows diffusion coefficients of single pEGFP-N1-32x RNAs in cells as measured by MB imaging. The high variance of diffusion coefficients among transcripts in both the nucleus and cytoplasm indicates a heterogeneous nature of RNA dynamics . On average, RNAs in the nucleus move nearly 4 times slower than transcripts in the cytoplasm, consistent with previous findings showing that the nucleoplasm is more viscous than the cytoplasm . Similar results have been obtained in cells transfected with pEGFP-C1-32x RNAs (Figure 4B), in which the 32 MB target repeats are located in the 3′-UTR. These findings suggest that an RNA transcript can be targeted by 2Me/PSLOOP MBs at either 5′- or 3′-UTR. Furthermore, binding of the 2Me/PSLOOP MBs to 32 tandem repeats does not cause interference with EGFP translation as seen when 64 repeats are used (Figure 4C), suggesting that MBs can be used to image target RNA containing up to 32 tandem repeats without perturbing physiological functions of target RNAs. There is no change in cell viability or cell spreading detected in cells microporated with varying concentrations of MBs (Figure 4D–E), suggesting that 2Me/PSLOOP MBs do not affect cellular growth or physiology. Overall, these findings suggest that 2Me/PSLOOP MBs can be a noninvasive platform for imaging single RNA transcripts in living cells.Figure 4Noninvasive imaging and measurement of RNA dynamics at the single-molecule levelSingle-particle tracking analysis reveals diffusion coefficients of single pEGFP-N1-32x (A) or pEGFP-C1-32x (B) mRNAs in the nucleus and the cytoplasm of HeLa cells. Insets show average diffusion coefficients (mean ± SE) in the nucleus and the cytoplasm. Significant difference is indicated with asterisks (P < 0.05). (C) The effect of 2Me/PSLOOP MBs on EGFP protein expression. Total EGFP protein level was assessed using Western blotting 24 h after microporation of 2Me/PSLOOP anti-repeat MBs at concentrations of 0, 1, or 5 µM into HeLa cells stably expressing pEGFP-N1-32x or pEGFP-N1-64x RNAs. Protein expression is normalized to that in cells microporated with 0 µM MB. Total GAPDH protein level was measured as a loading control. Significant difference from 0 µM MBs is indicated with asterisks (P < 0.05). Average spreading (D) and proliferation (E) 24 h after microporation with different concentrations of 2Me/PSLOOP MBs. All data are presented as mean ± SD of at least three independent experiments. The images are reproduced with permission from Elsevier .