See the abstract, outlining the purpose of this selection, here. See the first progress report here.
A total of five rounds of selection and a binding assay have been performed since the last progress report. mCherry, a monomeric fluorescent protein, has remained the target. The results for the first six rounds of selection, determined through the binding assay, were dismal – deeming the selection a failure. With such poor results, and discovering that mCherry has minor RNase activity, I determined it best to restart selection, utilizing tRNA to reduce background binding and perhaps limit any lingering RNase activity.
Initial Conditions (beginning at Round Four):
Beads to Use: Nickel-NTA
Incubation Time and Temperature: 20 minutes at 37 0C
Buffer and pH: PBS SELEX, pH 7.4
RNA:Protein Ratio: 400:200
Wash Volume and Number: 3 washes, 4 volumes
Negative selection was again performed. In an attempt to further reduce background binding, the RNA:protein ratio was reduced from 400:400 to 400:200. A decreased amount of protein would increase competition between the aptamers, hopefully singling out the stronger and/or faster binders.
Cycle course PCR was performed to determine the appropriate number of amplification cycles to be utilized in Large scale PCR (lsPCR). For Round Four, the DNA amplified much earlier than wash three (W3), with essentially no DNA present. As nine cycles appeared under-amplified, and twelve over, eleven cycles of the elution (E1) were determined for utilization in lsPCR. The gel can be seen in Figure 1. The remainder of the round progressed according to the protocol. The concentration of RNA resulting from PAGE and elution was 79.26 uM.
For Round Five, the previous conditions were kept the same, opting to perform a plus and minus selection. Although nothing was changed, the cycle course PCR revealed something seemingly unexpected: W3 + appeared in large amounts. As W3 had been seemingly non-existent in the previous two rounds, this was surprising. One hypothesis could be that the double pool binding reaction utilized, with only 40 uL of beads, contained many bead binders. Thus, when negative selection was performed, and the supernatant removed, perhaps many bead binders still remained, leaving many for the positive (with protein) portion. With essentially half as many beads to bind to, they were transferred into the positive portion of the selection. However, the exact cause of this is unknown. Amplified well, but not over-amplified, nine cycles was determined for utilization in lsPCR. The gel for ccPCR can be seen in Figure 2. The remainder of selection again progressed according to the protocol. The concentration of RNA resulting from PAGE and elution was 77.25 uM.
Round Six of selection returned to the initial conditions of Round Four, but with alterations to incubation times. RNA:protein incubation time was changed to fifteen minutes; incubation time during binding and selection was increased to four minutes, making the time between buffer removal and addition approximately five minutes. These alterations would address binding kinetics. The overall reduction in incubation time would hopefully allow faster binders to predominate; the increase in wash incubation time would potentially allow weak binders to fall off – potentially further isolating more mCherry-specific aptamers.
Cycle course PCR was again performed to determine the appropriate number of amplification cycles to be utilized in lsPCR. For Round Six, the elution amplified before the third wash, but the presence of the third was still disconcerting. In addition, the elution amplified much later than in previous rounds – double the cycles of the Round Five. What caused such a change exactly is unknown. A later amplification could suggest that specific strands were isolated, and the non-specific washed away, but the binding assay performed, following this round, suggested otherwise. The gel for Round Six can be seen in Figure 3. The remainder of this round adhered to the protocol. The concentration of RNA resulting from PAGE and elution was 20.02 uM. The cause of such a low yield is unknown, as the crush-soak elution protocol was executed as normal.
The bead-based binding assay, utilizing Rounds One, Three, Four, and Six, was performed mostly by Holli Duhon. “Hot” Transcription, “Hot” PAGE, and the elution were performed prior to my involvement, which began with ethanol precipitation. One “round” of Katherine Li’s was also utilized.
Ethanol precipitation was performed as normal, taking extra care when working with radioactivity. The samples were resuspended in 50 uL of diH2O. Each round was then read in the spectrophotometer – revealing very low recovery. With such a low yield, a ratio of 50 pmol RNA:50 pmol protein was utilized in the “Hot” Selection. Although I was again not present for this process, Ms. Duhon explained that the procedure was the same as a regular plus and minus protein selection (without negative selection), stopping after the washes and elution. Wash zero, wash one, wash three, and the elution, for RNA exposed to protein (plus) and not exposed to mCherry (minus), were all utilized. The rig utilized was loaded as outlined in Figure 4.
At this time, I do not have a hard copy of the results. However, after seeing the results and talking with Dr. Brad Hall, Holli Duhon, and Katherine Li (also present for the assay), the results were dismal. In addition, Austin Rezigh and Dr. Brad Hall discovered that all the fluorescent proteins, including mCherry, had RNase activity. Although Dr. Hall stated that most of the nucleases should have been removed while washing the immobilized protein with 400 uL of 1X PBS, three times, this is uncertain. Thus, I determined it was best to restart selection from Round One, utilizing tRNA to not only decrease background binding, but also combat any remaining RNase activity. This would hopefully be accomplished as the tRNA would provide competition during protein binding, and also serve as a buffer to nuclease activity. With more tRNA, 8X the amount of RNA (in pmol), there is a higher probability the nucleases would chew up the tRNA, over the N34 RNA pool.
Initial Conditions (Round One plus tRNA):
Beads to Use: Nickel-NTA
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
This first round adhered to the protocol. The tRNA, 3200 pmol at 315 uM (about 10.16 uL), was added when the pool binding reaction was added to the protein. Cycle course PCR was performed to determine the appropriate number of amplification cycles to be utilized in large scale PCR. For this first round, Wash Three and the Elution amplified at about the same time. As nine cycles appeared under-amplified, and twelve cycles over-amplified, eleven cycles was determined for utilization in large scale PCR. The gel can be seen in Figure 5. The concentration of RNA resulting from PAGE and elution was 80.84 uM.
To combat non-specific RNA, the RNA:protein ratio was reduced in the second round from 400:400 to 400:200. As stated previously, this again would hopefully provide competition between the RNA, with the faster and/or stronger aptamers binding to the decreased amount of protein. tRNA was again utilized for this round.
Cycle course PCR was again performed as stated. Although E1 amplified before W3, there was still a significant amount of amplification in the third wash. This will be addressed in future rounds. As nine cycles appeared well-amplified, but not over-amplified, it was utilized for large scale PCR. The gel can be seen in Figure 6. The concentration resulting from PAGE and elution was 105.13 uM.
In Round Three, and future rounds, promoting mCherry-specific binding and decreasing background binding will be of the highest priority. Steps will be taken toward this goal in Round Three, as the wash volume will be increased from 200 uL to 400 uL. In addition, negative selection will also be performed.