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(a) DNA scaffold logic using FRET signal cascades. (b) A fluorescence dye corresponding to site i of a DNA scaffold switches between the on and off states according to the presence of an input molecule (input ). (c) Configuration for the AND logic operation. Fluorescent molecules of a FRET pair are assigned to neighboring sites, site i and site j. (d) Configuration for the OR logic operation. Multiple input molecules can deliver a fluorescent molecule to a single site.
Designed secondary structures of (a) connecting and (b) disconnecting DNA strands during the absence or presence of the input molecule. C:connecting DNA strand; D: disconnecting DNA strand; I: input molecule; S: DNA scaffold with a binding site. RR and AR indicate the recognition and address regions. Kinetic fluorescence experiments of (c) connecting and (d)disconnecting DNA strands. The excitation wavelength was 496 nm, and emission intensity at 517 nm was measured. After recording the baseline signal produced by the FAM of C1 or D1, Sq and I1 were added at 500 s and 1000 s, respectively. The final concentrations of I1 and other components were 2 μM and 0.4 μM in phosphate buffered saline containing 30 mM NaCl (pH 7.4). Experiments were conducted at . The broken line indicates fluorescence change caused by dilution.
Circuit diagram and fluorescence outputs of (a) , (b), and (c) . Concentrations of the input molecules were 0 μM and 2 μM when the input values were “0” and “1.” The concentrations of the other components were 0.4 μM. The excitation wavelength was 470 nm and fluorescence spectra were measured at equilibrium after adding the inputs. The measured spectra were decomposed (see Ref. 23) to extract the emission of the reporting molecule. The value of the output fluorescence signal is defined as the difference between the intensities of the emission peak wavelengths of the reporting molecules with and without inputs.
(a) Circuit diagram and (b) fluorescence outputs of the three-input AND operation based on two-step FRET. Experimental conditions were the same as those in Fig. 3. (c) The number of fluorescent molecules consisting of FRET path, N, versus run-time of the DNA scaffold logic. Here, the response time is defined as the time to the half-completion of the output fluorescence intensity at equilibrium after adding the inputs. In this experiment, all final concentrations for operations were 0.4 μM in 50 μl of phosphate buffered saline containing 30 mM NaCl (pH 7.4).
DNA sequences and dye modifications.
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