Fig. 3: In-solution fluorescence measurement of finger domain movement.

a Structural illustration of finger domain movement responsible for fluorescence signal variation. The structures represent the finger domain in the closed state (CG complex, cyan) and the open state (Qtag-ins1 model predicted by AlphaFold, orange). Regions undergoing the largest conformational changes during finger closure---the O-helix, N-helix, and the intervening loop---are fully colored, while other segments are shown in a translucent view. The second glutamine in the Q-tag is depicted in red sphere, with the approximate corresponding location in the CG complex shown in blue sphere. The nucleobase modified with BHQ is colored black. b DNA sequence used for the fluorescence study with the BHQ-modified thymine highlighted in black. Note that the nucleobase marked in black in (panel a) indicates the position of the BHQ modification but does not correspond precisely to the modified nucleobase in the fluorescence experiments. The DNA sequence in the CG complex differs from that used in the fluorescence study. c Fluorescence emission spectra of FAM-labeled Qtag-ins1 bound to the BHQ-modified DNA with the correct incoming nucleotide dATP and ART558. The unit of fluorescence intensity, a.u., refers to arbitrary units. d Comparison of integrated emission peak intensities (510-530 nm) in the presence of 2 mM of each incoming nucleotide, illustrating the varying degrees of finger domain closure. The open and closed states are defined by the binary complex of the enzyme and DNA, and the ternary complex of the enzyme bound to DNA, dATP, and ART558, respectively. Data are presented as mean ± SD from four independent experiments. *p  <  0.1, **p  <  0.01, ***p  <  0.001, determined by a two-sided t-test. Paired for comparisons between dATP and dATP+ART558, and unpaired for comparisons of dATP with dCTP, dTTP, or dGTP. Source data are provided as a Source Data file.