SUPPLEMENTARY MATERIALS

Supplementary Figure S1. Emission spectra of non-PS-modified and PS-modified anti-luciferase MBs. 50 nM MBs (Cy5 fluorophore and Iowa Black® RQ-Sp quencher) was recorded in the absence (dash line) and presence (solid line) of excess complementary oligonucleotide targets. The addition of complementary target led to a large increase in MB fluorescence.

Supplementary Figure S2. Nonspecific opening of non-PS-modified and PS-modified MBs in living HEK293 cells. (A) Representative images of anti-luciferase MBs acquired at 10 h following microporation into HEK293 cells. The MBs used were not complementary to any known endogenous RNA or DNA sequence in 293 cells. The inset shows an expanded segment of the image. Arrows point to bright puncta in the nucleus. (B) The percentage of nonspecific opening was quantified over the course of 10 h as described in Materials and Methods. Note that 2Me/PSLOOP anti-luciferase MBs exhibit the lowest false-positive signals and were least susceptible to nonspecific opening. Each data point represents the mean ± S.E. from at least 40 cells. (Scale bar, 10µm)

Supplementary Figure S3. Nonspecific opening of non-PS-modified and PS-modified MBs in living Jurkat cells. (A) Representative images of anti-luciferase MBs acquired at 10 h following microporation into Jurkat cells. The MBs used were not complementary to any known endogenous RNA or DNA sequence in Jurkat cells. The inset shows an expanded segment of the image. Arrows point to bright puncta in the nucleus. (B) The percentage of nonspecific opening was quantified over the course of 10 h as described in Materials and Methods. Note that 2Me/PSLOOP anti-luciferase MBs exhibit the lowest false-positive signals and were least susceptible to nonspecific opening. Each data point represents the mean ± S.E. from at least 30 cells. (Scale bar, 10µm)

Supplementary Figure S4. Nonspecific opening of non-PS-modified and PS-modified MBs in living primary BJ cells. (A) Representative images of anti-luciferase MBs acquired at 10 h following microporation into BJ cells. The MBs used were not complementary to any known endogenous RNA or DNA sequence in BJ cells. The inset shows an expanded segment of the image. Arrows point to bright puncta in the nucleus. (B) The percentage of nonspecific opening was quantified over the course of 10 h as described in Materials and Methods. Note that 2Me/PSLOOP anti-luciferase MBs exhibit the lowest false-positive signals and were least susceptible to nonspecific opening. Each data point represents the mean ± S.E. from at least 30 cells. (Scale bar, 10µm)

Supplementary Figure S5. Emission spectra of non-PS-modified and PS-modified anti-repeat MBs. 50 nM MBs (ATTO647NN fluorophore and Iowa Black® RQ-Sp quencher) was recorded in the absence (dash line) and presence (solid

line) of excess complementary oligonucleotide targets. The addition of complementary target led to a large increase in MB fluorescence.

Supplementary Figure S6. Nonspecific opening of non-PS-modified and PS-modified anti-repeat MBs in living HeLa cells. (A) Representative images of anti-repeat MBs acquired at 10 h following microporation into HeLa cells. The MBs used were not complementary to any known endogenous RNA or DNA sequence in HeLa cells. The inset shows an expanded segment of the image. Arrows point to bright puncta in the nucleus. (B) The percentage of nonspecific opening was quantified over the course of 10 h as described in Materials and Methods. Note that 2Me/PSLOOP anti-repeat MBs exhibit the lowest false-positive signals and were least susceptible to nonspecific opening. Each data point represents the mean ± S.E. from at least 40 cells. (Scale bar, 10µm)

Supplementary Figure S7. Detection of single RNA transcripts using a lower concentration (1µM) of 2Me/PSLOOP and 2Me/PSFULL MBs. A) Colocalization analysis of MBs to smFISH signals. B) Colocalization analysis of smFISH to MB signals. Data represent mean ± SD of at least 10 cells. *represents sigificant differences from the 2Me/PSFULL MB.

Supplementary Figure S8. Detection of single RNA transcripts using 2Me/PSFULL

MBs and smFISH. After microporation of HeLa N1-32x cells with 2Me/PSFULL anti-repeat MBs, the cells were fixed and smFISH was performed to assess the accuracy of MBs for detecting single RNA transcripts. Representative maximum intensity projection images of 2Me/PSFULL MBs and EGFP smFISH at 8 h and 24 h post-microporation are shown. Quantification of colocalization between MBs and smFISH is shown in Figure 4. (Scale bar, 10µm)

Supplementary Figure S9. Representative images of cell spreading following microporation of 2Me/PSLOOP MBs at various concentrations. 2Me/PSLOOP MBs were microporated into HeLa cells at 0, 1 or 5µM. 24 h post-microporation cells were stained with Texas Red-C2-Maleimide. Non-microporated cells are shown for comparison. (Scale bar, 10µm)

Supplementary Table 1. The set of probes against the EGFP coding sequence used for smFISH imaging of individual engineered repeats. All of the probes were labeled with a TAMRA flurophore at the 3’end.

Supplementary Movie 1. Representative movie of individual mRNA transcripts moving within a cell. The cell is expressing pEGFP-N1-32x RNAs, which appear as discrete fluorescence spots.

Supplementary Movie 2. Representative movie of individual mRNA transcripts moving within a cell. The cell is expressing pEGFP-N1-32x RNAs, which appear as discrete fluorescence spots.

Supplementary Movie 3. Representative movie of individual mRNA transcripts moving within a multinucleated cell. The cell is expressing pEGFP-N1-32x RNAs, which appear as discrete fluorescence spots.

Zhao et al., Fig. S1.

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Zhao et al., Table S1.