Fig. 7: TH intron 10 folds into a parallel G4 with the potential to directly inhibit polymerase transcription.
a Coding strand sequence of the first 126 bp of TH intron 10 which surrounds the predicted G4. Grey blocks illustrate sequences of probes tested for G4 folding using CD. b CD spectra of fragment 4 (Frag 4 53-92), positive parallel G4 control Myc Pu22, and a poly dT negative control. Spectra are a average of 10 accumulations (c) relative ellipticity of fragment 4 with or without TMPyP4 at 263 nm and a temperature range of 20–95 °C. d DNA construct and primer design used for in vitro G4 polymerase inhibition assay. e PCR products amplified by the high fidelity Q5 polymerase. Final TMPyP4 concentration from left to right was 0, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM. f Quantification of product shown in (e). Band intensity was normalized to internal, vehicle control. Statistical analysis was performed using a two-way ANOVA with mixed-effects analysis and Dunnett’s test. p values represent TH + intron 10 at the respective TMPyP4 concentration compared back to 10-3 nM TMPyP4. Mean ± SEM. n = 4 independent replicates per condition. g DNA construct design used for in vivo G4 inhibition assay. h mCherry amplification 60 h post-transfection and treated with 1.25 µM TMPyP4. Data is normalized to time-matched, untreated controls. Mean ± SEM. Statistical analysis was performed using an unpaired two-tailed t-test. For statistical analysis of the entire dose response curve see Supplementary Fig. 13c and source data. n = 3 independent replicates per condition. i Proposed model of DSR regulation.
