Progress: EphA2 selection and database

Click to view the full proposal and the abstract.

R6 of selection on EphA2 using N34 pool was carried out using bead-based negative selection. The RNA:EphA2 ratio was reduced from 200 pmol RNA:200 pmol EphA2 to 200 pmol RNA:100 pmol EphA2 in order to reduce the amount of high concentration of background binders indicated by the binding assay. 11 rounds of lsPCR were performed to amplify the DNA as shown in figure 1. The final concentration of R6 is 49.01 uM.

R7 of selection on EphA2 was carried out using bead-based negative selection. The RNA was allowed to incubate with the beads for 35 minutes, as opposed to 30 minutes, in order to increase the amount of background binders onto the beads. The wash volume was also increased to 4 wash volumes as opposed to 2 wash volumes in order to increase stringency and the presence of binding aptamers to the protein. However, as ccPCR indicated, the conditions may have been too stringent as the only band present was in C20, as indicated in figure 1. Selection on this round was not continued in order to begin work on the aptamer database.

Figure 1 ccPCR gels. 3.8% agarose gel was used to separate the amplified DNA products of R6 and R7. 11 rounds of PCR were sufficient to amplify the DNA in R6; however, when selection conditions increased in stringency, the DNA was amplified after 20 rounds of PCR. The more rounds of PCR needed to amplify the DNA indicates a decrease in the amount of background binders and decreasing variability in the pool, highlighting the potential impact of aptamers on the medical world.

64 articles regarding the original selection of various aptamers, along with unique selection techniques, were inputted into Mendeley. The targets varied greatly from multiple HIV related proteins to anti-aging related proteins showing the wide range of uses for aptamers.

Because the binding assay indicated that there was a low amount of binders after 5 rounds of selection, a new selection was begun. To follow the experimental conditions of Sharon Reimann, a 2008 student who achieved ~10% binding after 5 rounds of selection using the N35 pool and EphA2 through filter based selection, filter based selection on the N34 pool and EphA2 was begun today. HEPES selex will be used as the binding buffer and the protein+RNA bound on the filter will be washed with 1 wash volume 3 times. Incubation time is 25 min at 25C. 400 pmol N34 RNA was incubated with 200 pmol EphA2. In future rounds, the RNA:EphA2 ratio will decrease to 200 pmol RNA:200 pmol EphA2.Selection will continue to be performed under the same conditions until R3 when negative selection will begin. After 5 rounds of selection, a binding assay will be performed in order to determine the binding affinity of the remaining nucleic acids.

1 comment:

Brad Hall said...

can you repost this figure. It didn't show up well.