Fig. 1: Characterization of CssDNA and regulation of its protein expression in the CFE system.

a Design schematics of CssDNA vectors, where colors depict positions of vector features such as T7 promoter (red), EGFP (green) and M13 ori (orange). In sense circular single-stranded DNA (CssDNA(+), left), the coding sequence of the expression cassette is presented from 5’ to 3’. In antisense circular single-stranded DNA (CssDNA(−), right), the template sequence of the expression cassette is in the reverse direction. b 1% agarose gel analysis of CssDNA(+) and its corresponding plasmid (S-plasmid), CssDNA(−) and its corresponding plasmid (AS-plasmid). c AFM images of CssDNA (left) and plasmid (right), scalebar 200 μm. d Schematic of gene expression of CssDNA(+) or CssDNA(−) vector in the CFE system. The CFE system contains all the essential components for CssDNA gene expression, with some representative components (not all) shown in the dashed box. e, f Changes in fluorescence signal over time for CssDNA(+) (e) and CssDNA(−) (f) vectors (5 ng/μL) during expression relative to the blank group. g Schematic representation of the regulation of CssDNA(+) or CssDNA(−) vector gene expression by dNTPs and aphidicolin in the CFE system. h, i Changes in protein expression levels over time for CssDNA(+) (h) and CssDNA(−) (i) vectors (5 ng/μL) in the presence of dNTPs or aphidicolin. All fluorescence signals were normalized according to the fluorescence intensity of the highest expression level of the corresponding expression vector. The maximum rate constant of the fluorescence kinetic curves in e, f, h and i was obtained by taking the first derivative of the corresponding curve, as shown in Supplementary Fig. 4. Data collected in e, f, h and i were monitored by a microplate reader and are presented as mean ± standard deviation (s.d.) for n = 3 biologically independent experiments, source data provided.