File:A-microRNA-initiated-DNAzyme-motor-operating-in-living-cells-ncomms14378-s2.ogv

English: Supplementary Movie 1a Imaging of fluorescence of Cy5 from individual AuNPs. Fluorescence images demonstrate operation of the DNAzyme motor on individual AuNPs in the presence of target miRNA sequence. The white dots on black background are due to fluorescent Cy5 on the AuNPs as depicted in Supplementary Fig. 14. The increases in fluorescence over time represent the miRNA-initiated stepwise walking of the DNAzyme motor on the AuNP and the corresponding catalytic cleavage of the quencher-labeled substrate. Forty five microliters of the operating solutions contained either 300 pM target miRNA, the DNAzyme motor (Supplementary Fig. 14) at an equivalent concentration of 30 pM functionalized AuNP, 25 mM Tris-acetate buffer (pH 8.0), and 200 mM NaCl. After incubation at room temperature for 20 min, 0.5 mM MnCl2 solution was added to initiate the operation of the motor. The time 0 min in the figure refers to when the MnCl2 was added. One μL of the incubation mixture was transferred to a microscope slide (25 –76 –1.0 mm; Fisher). A micro coverglass (diameter: 18 mm, thickness: 0.16-0.19 mm; Electron Microscopy Sciences) was placed over the solution on top of the slide. The AuNPs sandwiched between the microscope slide and the coverglass were then imaged using a DeltaVision OMX Imaging System (GE Healthcare Life Sciences). A 60X/1.49 TIRF objective (Nikon) was used, and a 647-nm laser provided excitation. One hundred and twenty frames were acquired in 30 min with 10% laser power and 100 ms exposure time for each frame.