Part:BBa_K4245135:Experience

The Lettuce split aptamer has been tested with the complement sequence of the middle sequence (BBa_K4245131) as well as with RCP produced through Rolling Circle Amplification (RCA).

We attempted to simulate the binding of the split Lettuce to the RCP by testing the split Lettuce with a sequence complementary to the middle sequence ordered from Integrated DNA Technologies. The complement, split Lettuce, and DFHBI-1T were mixed and heated at 70°C for 5 minutes, then cooled and held at 41°C for 1 hour. The fluorescence before and after were measured on the plate reader. From the results, we saw an increase in fluorescence in the presence of the complementary middle sequence and lettuce in the reaction as compared to the controls (see Fig. 1). This suggests that the RCP produced through this middle sequence would allow for successful hybridization between the split Lettuce and RCP.

Figure 1. Graph of split Lettuce reaction with middle sequence complement.

RCA was run with hsa-miR-1-3p RCA Padlock Probe BBa_K4245200. The products, split Lettuce, and DFHBI-1T were mixed and heated at 70°C for 5 minutes, then cooled and held at 41°C for 1 hour. The fluorescence before and after were measured on the plate reader. (see Fig. 2)

Figure 2. Graph of split Lettuce reaction with RCP. The values represent the change in fluorescence before and after the reaction with DFHBI-1T took place.

The increase in fluorescence of the RCP + Lettuce + dye was significantly greater than the controls, which suggests that the split Lettuce was successfully bound to the RCP and induced fluorescence in DFHBI-1T.

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