Fig. 2: Transcription in the inner IDP droplet.

a Schematic of transcription in the inner IDP droplet. Enzymes (RNAP, RNA polymerase) and repressors (LacR) are both designed to bind with IDP through SZ1-SZ2 peptide interactions. LacR binds to the lacO sequence placed outside the region to be transcribed, thus recruiting template DNA to the IDP droplet. Transcribed RNA contains a binding site for a molecular beacon (MB) for fluorescence observation. b, c Enrichment of SZ2-tagged RNAP confirmed by fluorescent labeling by fluorescein (FL). Line profiles were extracted from white dashed lines. White bold scale bars at lower right represent 5 µm (b). Exact p-values are 2.6e-14 (SZ2-T7RNAP-FL) and 3.0e-9 (T7RNAP-FL) (c). The experiment was repeated at least three times with similar results. d, e Enrichment of fluorescence-labeled lacO-containing DNA in the presence or absence of SZ2-LacR. Line profiles were extracted from white dashed lines. White bold scale bars at lower right represent 5 µm (d). Exact p-values are 1.8e-13 (FL-lacO DNA + SZ2-LacR) and 6.6e-52 (FL-lacO DNA) (e). The experiment was repeated over three times with similar results. f Time-lapse images of transcription monitored by MB. The scale bar represents 10 µm. See also Supplementary Fig. 7a for line profiles. g Fluorescence intensity of MB was measured in the outer Dex region of droplet-in-droplet structures to quantify transcription activity. Data are presented as mean values ± 2SE. Under each experimental condition, over 250 droplet-in-droplet structures were imaged across four distinct microscopic fields per frame. Significance was assessed using a two-tailed Welch’s t-test: NS (p > 0.05), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).