Progress Report 2 on mTagBFP RNA Bead-Based Selection

To gain a general understanding as to the purpose and direction of this selection, here is the target abstract.

Thus far, five rounds of RNA bead-based selection have been performed on mTagBFP, a fluorescent protein emitting a blue color. Overall, the selection is progressing well. However, I fear that actual binding to my protein is very insignificant. While I have not yet performed binding assay, justification for this concern will presented here after.
Initial Conditions for Round 3 of selection were as follows:

Target: mTagBFP
Beads to Use: Nickel-NTA
Pool: N34
Incubation Time and Temperature: 20 minutes at 37 0C
Buffer and pH: PBS SELEX, pH 7.4
Ratio: 400 pmol RNA: 400 pmol mTagBFP
Wash Volume and Number: 4 volumes (400uL), 3 washes

Note: These conditions are similar to those of Alec Rezigh as our research is partnered.

For round 3, a negative selection was performed in the hope of reducing the number of background binders. Following reverse transcription (RT), the wash zero (W0), wash three (W3), and elution (E1) samples collected during cycle course PCR (ccPCR) were run down a agarose gel. As can be seen from Figure 1 below, the elution sample was slightly overamplified at cycle twelve, but under amplified at cycle nine. Therefore, eleven cycles were chosen for the amplification of the elution ssDNA during large scale PCR (lsPCR).



Still promising thus far is the non-observance of W3 samples. This could indicate significant protein binding but likely suggests the presence of immense background binding. Again however, this cannot be determined definitively until a binding assay is performed.

With the ultimate goal of isolating a strong binding aptamer, a reduction in the protein to RNA ratio from 400:400 to 200:400 was implemented to hopefully select from stronger binders and weed out weaker ones. After conducting ccPCR on this rounds W0, W3, and E1 RT products, the ccPCR results were as follows:



As was observed again, the E1 sample was underamplified at 9 cycles but overamplified at 12 cycles. Consequently, 11 cycles was determined to be the optimal number of cycles for lsPCR. Interestingly, the previously invisible W3 samples appeared in this round. However, this is somewhat expected due to the reduction in the amount of protein and thus area in which the pool can bind.

At round 5, a plus/minus (+/-) selection was performed. Running the W0 and E1 plus and minus protein samples (W0(+), W0(-),E1(+), E1(-)) down a gel, the results were as follows:



As can be seen, E1(-) amplified before E1(+). This is greatly undesirable as it suggests significant background binding or target RNase activity. While a binding assay will have to be performed to understand the extent of this background binding, an RNase Alert test (kit provided by Ambion and instruction from Dr. Brad Hall) was performed to examine if my protein exhibited nuclease activity. While the data for this test will be provided in a separate post, my protein did in fact exhibit RNase activity; approximately 10% of the positive control over a 30 minute time period.

The concentrations yielded from round three, four and five were 75.59uM, 53.05uM, and 42.66uM, respectively.

Overall, the selection process against mTagBFP has been satisfactory. I hope to eliminate small as well as large personal errors to increase the speed and accuracy of each round. To hopefully reduce background binding and stop some of my pool from being chewed up by mTagBFP’s nuclease activity, tRNA will be utilized. Once the next round is complete, a binding assay will be performed. The results of this assay will decide the future of this selection.