Progress: RNA Selection (Beta Lactamase)

The full proposal has been posted on Dropbox, the abstract is here.

Progress, Results, and Discussion

Two rounds of bead based selection with N34 RNA against Type IV penicillinase have been completed. Below are the gel images of the ccPCR E1 and W3 from selection. Large scale PCR was performed at 12 cycles for R1 and 9 cycles for R2. In R2, the elution amplified earlier than in R1, possibly signifying an increase in binders. However, these binders may not actually bind the target. They could be binding the beads or other components in selection.

As shown in Figure 1, the elution in R2 amplifies earlier than in Round 1. This could be an increase in binding sequences, but the more likely explanation is an inadequate wash volume. The gel image of W3 below (Figure 2) shows that there are many nucleic acid sequences that are not being washed away effectively.

Figure 2 shows that a large portion of the pool is still not being separated from the protein through consecutive washes. To resolve this problem, the wash volume will be increased in the next round. In addition, negative selection will be performed in R3, in order to reduce background binders. Finally, the incubation time between washes will be lengthened in order to allow all of the nonbinders to wash away from the target.

The concentrations at the end of R1 and R2 were 58.6 uM and 100.9 uM, respectively. This increase in concentration is a good sign, as it may indicate an increase in the concentration of binding species.

Problems Encountered

There were no outstanding problems in either R1 or R2. As outlined above, the wash volume and incubation times between removal of selection buffer will be increased in order to solve the problem of nonbinding sequences in washes.

Conclusion and Future Work

Two successful rounds of bead based selection against beta lactamase have been completed. The concentration at the end of R1 was 58.6 uM, and 100.9 uM for R2. By the next reporting period, R3 and R4 will have been completed. Negative selection will have started to separate those sequences that bind components of selection rather than the target. Progress results will be posted following subsequent rounds of selection.