Six rounds of bead based selection (using Streptavidin beads) have been performed with N34 RNA on beta-lactamase (Type IV penicillinase) using 400 pmol RNA each round; 10X HEPES SELEX buffer was used to wash away nonbinding species. The protein/RNA was incubated at 37C for 25 minutes. Protein:RNA ratios and wash volume/number were dependent upon the round number. This information is in Table 1.
Since the last reporting period, four additional rounds have been completed, for a total of six rounds. Gel images from ccPCR give great insight about the selection process. Shown below in Figure 1 is an image of the last wash from each of the six rounds. Despite repeated attempts to decrease nonbinding species in the last wash, these sequences seem to stick to either the target or the beads. Wash number and volume were each increased, in addition to an increase in incubation time between washes. None of these provided any positive results, as amplification occurs at around the same number of cycles each round.
An image of the elution for R1-R6 can be found in Figure 2. Cycles in which optimal amplification occurs are boxed in. These are the number of cycles to be used in lsPCR. Amplification occurs unusually early, especially in R5. This could be cause for concern, as background binders could be present in the elution. However, this cannot be conclusively stated without binding assay data. In spite of this, most of the elution shows up before the washes.
Negative selection was performed in R3, R5, and R6 in order to minimize the sequences that bind the beads. tRNA was used in R6 negative selection so that beta-lactamase-binding sequences are not degraded by possible RNAse activity.
Even though numerous measures were implemented to decrease sequences in the last wash, there is still a high concentration of nucleic acid in this wash. This means that some of the nonbinding sequences carried over into the elution. A binding assay will need to be performed in order to truly determine this.
Conclusion and Future Work
Consequently, six rounds of bead based selection have been performed. A summary of this is shown below in Table 1. In order to quantitatively determine the binding affinity of isolated sequences for beta lactamase, a binding assay will be performed. This data will be posted shortly.