Since the last progress report, selection has been restarted due to the low binding as seen in the binding assay performed in August. Selection was restarted using filter based selection rather than bead based selection in order to prevent against inability for the protein EphA2 to adhere to the beads in bead-based selection. In contrast with the results from bead-based selection as seen in figure 1, the RNA pool is amplifying at earlier cycles during cycle course PCR, indicating that the pool is less variant and narrowing down to more specific binders.
Figure 1 ccPCR gels. These are the ccPCR gels from the previous round of bead-based selection. There is little indication of the decreasing variance of the pool, which would be indicated by earlier amplification.
Figure 2 ccPCR gels: The aliquots from the R1-R5 cycle course PCR products were separated through a 3.8% agarose gel at 125V for 25 minutes in order to determine the necessary cycles for large scale PCR. R1 required 15 cycles of PCR for sufficient amplification, R2 required 9, R3 required 12, R4 required 9 and R5 required 6.
The initial conditions for selection in R1 can be found in the previous progress report. R1 RNA was adequately amplified after 15 rounds of PCR, as seen in Figure 2 above. After transcription and purification, 51.7 uM RNA was isolated. Selection stringency was increased for round 2, as seen below.
Conditions for selection:
Incubation time and temperature: 25 minutes at 25C
Buffer and pH: HEPES SELEX, pH 7.5
RNA:Protein ratio: 200:100
Wash volume and number: 1 wash volume 1X HEPES SELEX buffer 3X
After decreasing the amount of protein available for the RNA to bind to, the necessary cycles for sufficient amplification was 9 cycles, indicating that the RNA pool is narrowing down in sequences and allowing the pool to amplify at an earlier cycle. R2 produced 48.5 uM RNA. Because of the earlier cycle necessary for amplification, selection conditions became more stringent. Negative selection was performed during R3 while selection conditions remained the same as R2. As seen in Figure 2 above, the required cycles for amplification was 12 cycles. R3 produced a final 67.5 uM RNA.
Negative selection was continued during R4. However, in order to increase the stringency, the initial binding reaction sans protein was allowed to incubate with the filter for five minutes in order to select against sequences that had weak binding affinity to the filter. The incubation time between the unbound RNA and the protein was also increased to 30 minutes at 25C to encourage binding between the protein and weaker RNA binders that did not bind to the filter. Also, the wash volume was also increased to 1.5 wash volumes 3X in order to select against negative binders. As seen in Figure 2, the necessary number of cycles needed to amplify the DNA was 9. Because of the increased stringency during this round, it can be assumed that earlier amplification cycle indicates the narrowing down of sequences that have higher binding affinity for the protein, or unfortunately, the filter. The final RNA concentration for R4 was 57.56 uM.
Because of the earlier amplification cycle, selection conditions became more stringent. The only difference from R4 was the increase in the number of washes from 3X with 1.5 wash volumes to 5X with 1.5 wash volumes. The increased stringency proved sufficient to further decrease the pool variance because the necessary cycles for amplification were 6. However, this amplification at such an early cycle produces some concerns. Although it may indicate the presence of strong binders to the protein, it may also indicate the selection of high affinity binders to the filter. Because of the increased stringency at early rounds, it may be possible that potentially strong binders to the protein were selected against and high affinity negative binders took over the pool, producing the early amplification in the cycle courses. Although the negative selections are aimed to prevent negative selection, it is unsure whether the DNA being amplified is binding to the protein or the filter. A binding assay is currently being performed in order to determine which of the aforementioned concerns are the likely causes for the early amplification trends in this round of selection.
As seen in Figure 2, there are two bands in the gel. This is most likely due to the formation of primer dimers which reduce the amount of actual product being amplified. Ways to decrease primer dimer formation are by altering the primer concentration, salt concentrations, and temperature of the reaction. By altering these factors, the actual amount of higher affinity nucleic acids being amplified during large scale can increase and subsequently increase the amount of nucleic acid PAGE purified.
Conclusion and future work
As compared to my results from bead-based selection, filter-based selection of the N34 RNA pool against the protein target EphA2 has been more successful. A binding assay is currently being performed in order to evaluate the actual binding affinity of the RNA to the target. From the binding assay results, further measures, such as increasing stringency, will be implemented to combat against negative binders and increase the selection of a high affinity binder to EphA2.