Fig. 1: Characterizing RNAh technology.

a, Mechanism of action of RNAh. rG4 induced by RNAh technology inhibits ribosome scanning, resulting in translational suppression. b, rG4 models have four G-tracts that are split into a 2 + 2 or 3 + 1 arrangement by 63-, 100- or 140-nt-long loops. c, Nucleotide sequences of rG4 models, Staple oligomer and Hybridization oligomer. G-tracts are shown in red. The dashed and solid underlines represent the 26-nt or 40-nt Staple oligomer recognition sites. d, Evaluation of rG4 formation with NMM. The blue and red curves show the fluorescence emission spectra of NMM in the presence of 26-nt or 40-nt DNA Staple oligomers, respectively. The solid and dashed curves represent the model sequence with G-tracts and the control sequence with A-tracts, respectively. e, Thermal denaturation profiles of Staple oligomer hybrids. An RNA strand containing either the 2 + 2–100-nt or the 1 + 1–100-nt sequence was complexed with a 5′-end FAM-labelled Staple strand, and the fluorescence intensity of the samples was measured from 20 °C to 100 °C. The graph displays the negative first derivative of the melting curve data (−dF/dT) versus temperature. Thermal melting curves of 2 + 2–100-nt and 1 + 1–100-nt sequences are shown in black and red, respectively. f, Identification of sequencing spectra on 2 + 2–100-nt and 3 + 1–100-nt rG4 model sequences by the RTase stop assay. The red arrowheads indicate the arrest site of RTase with rG4 induced by the Staple oligomers,and black arrowheads indicate the RTase elongation end without (w/o) rG4 induction. To indicate the location of the stop products, all sequencing spectra are represented below each specific sequencing spectrum. g, Evaluation of the effect of nucleotide length of DNA Staple oligomers on rG4 induction by the RTase stop assay. h, Dual reporter genes encoding FL and RL in tandem were used to validate the mechanism of action of RNAh. rG4 model sequence was placed in the 5′ UTR of FL. RL was placed downstream of an IRES as an internal standard. Staple oligomers induce rG4 formation (top), whereas hybridization oligomers do not (bottom). i, The effect of a 40-nt DNA Staple oligomer on the translation of reporter RNA in vitro. Data are mean ± s.d. of three independent experiments.