Fig. 2

Transcribing within the evanescent field. a–d Different DNA template labeling schemes result in different fluorescence intensity profiles. The DNA template labeling schemes are shown at the top of each trace with arrows showing the movement of the corresponding dyes towards or away from the surface during transcription progression resulting into a fluorescence intensity increase or decrease, respectively. The times of NTP injection and of labeled DNA oligo binding are indicated with arrows. The DNA oligo binds to the 3′-end of the nascent RNA and thereby scores for full-length RNA as illustrated in Fig. 1. Note that the characteristic fluorescence intensity changes also allow using a single color for monitoring transcription progression and scoring of full-length RNA with the labeled DNA oligo, i.e. Cy3 in (c, d). 532 nm and 642 nm lasers were used to excite the Cy3 and Cy5 dyes, respectively (a, c, d). A single laser at 532 nm was used to excite both the Cy3 and Cy3.5 dyes (b). e The total fluorescence intensity change during transcription, ΔI(TK), is related to the dye position change along the evanescent field. ΔI(diss) denotes the intensity change upon DNA template dissociation. f The absolute fluorescence intensity change during transcription widely varies between transcribed RNA molecules of the same size due to inhomogeneous illumination intensity across the ZMW chip. g Normalizing the intensity change during transcription by the intensity change during DNA template dissociation results in a nearly constant value (slope of curve) for all the molecules of the same length. h The normalized intensity change during transcription relates to the transcribed RNA length by a model in which the DNA template moves within the evanescent field (see Supplementary Fig. 2 and Supplementary Note 2). For (f, g), a 517 nt RNA was transcribed from a DNA template labeled as shown in (e); Number of molecules analyzed (n) = 83 (f, g) and (n) = 109, 65, 78, 72, 53, 70, 83 for the data points at 171, 199, 247, 388, 517, 517 and 517 nts transcribed RNA lengths (h). Source data are provided as a Source Data file