Fig. 5: RG4 does not recruit ribosome or increase RBS accessibility.

a–d Hypothesis one: RNA G-quadruplex (RG4) increases affinity to ribosome. a Schematic of RG4 recruiting the ribosome. b Competitor RNA used in translation reaction. The RG4 is cMyc (Fig. 3a), and the hairpin is 10 hp (Fig. 4a). c Normalized transcription rate of cMyc Non-template (cMyc-NT) with addition of competitor RNA. d Normalized translation efficiency. Additions of RNA 1–4 show weakly significant difference from cMyc-NT while RBS containing RNA (5 and 6) reduces GFP production, suggesting successful competition requires RBS not RG4. For (c) and (d), data are presented as mean ± SEM of independent experiments (n = 3). Exact mean values are provided in Supplementary Tables 3.4 and 3.5. Raw data points are provided as a Source Data file. Shown in (c, d) represents the significance between cMyc-NT and addition of competitor, where *P < 0.05, **P < 0.005 (two-sided paired t-test). e–g Hypothesis two: RG4 enhances the accessibility of RBS to ribosome. e Cartoon depicting the release of RBS by RG4. f Schematic for examining the accessibility of RBS. The mRNA is either heated to remove all secondary structure or not heated. A Cy5-labled molecular beacon complementary to the RBS is applied to determine accessibility. g Binding curves of the RBS molecular beacon to RNAs. The dissociation constants, K_d, are 367 (±25), 364 (±23), 370 (±21) nM for Non-Template (blue rectangle), Control (gray circle), and Template (purple diamond), respectively, showing a negligible difference in binding affinity (two-sided paired t-test). The dot curves shown are one representative result from n = 5 independent experiments. Raw data points are provided as a Source Data file. h Fluorescence intensities of molecular beacon with and without heating. There is no significant difference among the control, T, and NT with or without heating. Data are presented as mean ± SEM of independent experiments (n = 3). Exact mean values are provided in Supplementary Table 3.6. Raw data points are provided as a Source Data file. NS nonsignificant (two-sided paired t test).