FRET targeting experiments of ATTT and AAAA guide target combinations

100 pM of target construct annealed with biotin handle were flushed in the microfluidic chamber. After incubation of 1 min, the microfluidic chamber was rinsed with 100 μl T50 buffer. 10 nM of apo-CbAgo was loaded with 1 nM of ATTT seed DNA guide or with AAAA seed DNA guide at 37°C for 30 min in imaging buffer after which the mixture is introduced inside the microfluidic chamber. Movies of 200 s were taken at continuous illumination of the Cy3 signal. Site specific protein target interactions were identified as FRET signals and were further analyzed.