Progress Report of 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, two 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 personal errors have presented some hindrance.
Initial Conditions for selection were as follows:

Target: mTagBFP
Beads to Use: Nickel-NTA
Pool: N34
Incubation Time and Temperature: 25 minutes at 37 0C
Buffer and pH: PBS SELEX, pH 7.4
Ratio: 400 pmol RNA: 400 pmol mTagBFP
Wash Volume and Number: 2 volumes (200uL), 3 washes

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

For round 1, cycle course PCR (ccPCR) was performed on the reverse transcription products of wash zero (W0), wash three (W3), and elution (E1): samples acquired during selection. As can be seen from Figure 1 below, 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).



Promising in the gel was the fact that no samples showed up in the W3 portion of the gel. This could indicate either significant protein binding or immense background binding. However, this cannot be determined until a binding assay is performed in a few more rounds.

In the hopes of eliminating background binding, the wash volume for round 2 was increased from two (200uL) to four (400uL) volumes. After leaving out my RNA yield from the previous round for four days, reverse transcription of this product, along with W0, W3, and E1, was performed. After conducting ccPCR on all four products, the hope was that the round 1 RNA product would amplify far before the elution, illustrating that the RNA was not degraded. The ccPCR results were as follows:




As can be seen, W0 and W3 samples are missing for the large 3.8% agarose gel. Not shown as well are the 24 samples below the ones presented as they belonged to another student and were not visible either. Since one of the 100 base pair ladders showed up and one of them did not, it was concluded, with the help of Nia, that either there was not enough ethidium bromide present in the gel, or that there was enough, but it was not adequately dispersed throughout the gel to be able to visualize all of the samples. However, through a stroke of luck, the samples that showed up were the ones necessary to continue with the round. By looking at the gel, the RNA product was greatly overamplified at cycle six, demonstrating that the round 1 RNA yield was not degraded. Additionally, the elution sample at cycle twelve was adequately amplified, and thus was chosen for lsPCR.

The concentrations yielded from round one and two were 56.22uM and 58.92 uM, respectively. Due to the similarity between the two yields, further rounds of selection will have to be completed to make any conclusion as to whether mTagBFP aptamers are being amplified.

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. In next round, a negative selection will be performed and a decrease in incubation time from twenty-five to twenty minutes will be implemented in the hope of again eliminating bead/background binders as well as weak binders. In the future, other initial conditions will be modified to help isolate the best aptamer.