mCherry Fluorescent Protein Progress Report

See the abstract, outlining the purpose of this selection, here.

Three rounds of N34 RNA bead-based selection have been performed against mCherry, a monomeric fluorescent protein. The results so far have been promising, encountering no problems thus far.

Initial Conditions:

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

Cycle course PCR was performed to determine the appropriate number of amplification cycles to be utilized in Large scale PCR (lsPCR). For Round One, the DNA amplified around the same time in both wash three (W3) and the elution (E1), as seen in Figure 1. Amplified well, but not over-amplified, fifteen cycles was determined for utilization in lsPCR.

To combat this excess non-specifc DNA, the wash volume was increased from 200 uL to 400 uL. Upon visualization of the gel, the DNA in wash three was reduced significantly, while the DNA present in the elution increased. This could indicate augmented protein binding, but could also signify increased background binding. There is no evidence to support either, however. The cycle course from Round Two can be seen in Figure 2.

To continue reducing background binding, negative selection was performed in Round Three. The incubation time was also reduced from twenty-five to twenty minutes, hopefully weeding out any weak or non-specific binding. The results yielded a decrease in wash volume DNA and an increase in elution DNA, hopefully indicating an increase in protein binding. The gel can be seen in Figure 3.

The concentrations gained from Rounds One, Two, and Three, were as follows: 84.17 uM, 102.34 uM, and 107.15 uM. Ideally, this increase in concentration signifies the increased amplification of mCherry specific RNA. A binding assay after further rounds of selection will be utilized to determine this.

In Round 4, plus and minus selection will be performed to hopefully determine if an increase in protein binding is present. Other conditions, such as incubation time, RNA:protein ratio, and wash volume will be adjusted in future rounds to potentially isolate strong protein binding aptamers. A binding assay will be performed, after Round Five or Six, to check the progress and success of the selection.