Santiago Diaz Target: Myostatin
Progress, Results and Discussion
As the project advanced, many turns had to be taken. Firstly, the bead-based SELEX method had to be discarded due to the fact that the available myostatin was not biotinylated. Biotinylation of the protein would have been attempted if the concentration of protein solution and the amount of picomoles contained weren't so small. Therefore, it was decided that proceeding with filter-based SELEX was the best option. I began the procedure by preparing a target binding reaction between R0 N58 RNA pool and myostatin. The reaction was incubated for 25 minutes at a temperature of 37 ̊C. This reaction was then filtered using nitrocellulose filter paper to separate the bound species from the unbound pool. The nitrocellulose paper allows small molecules such as N58 RNA strands to flow through, and keeps larger molecules, such as proteins, on its surface. Consequently, the RNA strands that bind to the target stay on the filter paper, giving us the opportunity of retrieving those species by an elution process. After washing the filter paper with selection buffer three times, which is done in order to make sure that all the unbound species and weak binders flow through, the filter paper was placed inside a tube with elution buffer, which contains urea, an effective denaturant. I proceeded by heating the solution to 100 ̊C for 5 minutes, vortexing it, and exposing it to cold temperatures in order to further denature the RNA strands and pull them away from the target proteins. The solution containing the aptamers was taken out and placed on a new tube, labeled E1.
It was decided to run reverse transcription on E1 (selected aptamers). In order to run the reverse transcription, though, first I had to isolate the species. Therefore, I ethanol precipitated them, a process that brings RNA out of solution by forcing it to form a solid pellet. After removing the supernatants and resuspending the pure RNA species, I ran reverse transcription in order to transform all the selected and unbound RNA into DNA. This step is necessary if I wanted to get a decent amount of aptamer molecules from this process. Only an extremely low portion of the RNA pool actually binds to the target molecule when performing the binding reaction. But if I wanted the aptamer to serve its purpose, I needed much more than what was gotten from the random selection. Therefore, PCR (Polymerase Chain Reaction), a process that is capable of amplifying a desired strand of polynucleotides, had to be performed. However, PCR only works for DNA, as there is no RNA primer in existence that can withstand the high temperatures required for this process. For that same reason I had the selected RNA reverse transcribed into DNA.
Before proceeding to a large scale PCR, a cycle course PCR had to be run in order to determine the ideal number of PCR cycles needed to optimize correct amplification. Too few PCR cycles can't make a large enough functional amount of aptamers. On the other hand, too many cycles encourage over-amplification, which promotes the amplification of undesired DNA strands along with the selected one. The DNA bands corresponding to E1, obtained by using 3.8% agarose gel electrophoresis of Cycle Course PCR products, were going to be used to determine the ideal number of PCR cycles to which the large scale PCR should be run. To my surprise, there was almost no detectable amplification whatsoever, as shown in Figure 1. Besides the ladder, the only band that appeared was the one that corresponded to 20 cycles of PCR. This result is plausible, as the intial RNA-to-protein ratio was as small as 100pmol:100pmol. In addition, the base pair length of the obtained N58 dsDNA band (130bp) was very close to the real value (120bp). However, it was uncertain if the band represented the amplification of the actual aptamer or of other undesired strands, due to the lack of the No Template Control that I forgot to run.
Figure 1. Cycle course products gel electrophoresis. The only band that showed detectable amplification was Cycle 20 sample.
To make up for this, I decided to run the reverse transcribed E1 ssDNA, along with a “No Template Control”, for 20 cycles of PCR. The PCR products were ran through another gel electrophoresis, as shown in Figure 2. All the lsPCR products produced bands of equal size (130bp), along with some artifacts and other signs of over amplification. Fortunately, the “No Template Control” showed no band, thing that could be used as evidence to rule out the presence of foreign DNA. After seeing these positive results, I decided to proceed with the round of selection.
Figure 2. Large scale PCR products gel electrophoresis. All bands showed equal amplification, except NTC, which showed no band.
After isolating the lsPCR products via ethanol precipitation, transcription was performed on the obtained dsDNA. This step allowed me to transform the DNA into the originally selected RNA aptamers. This time, however, the number of aptamers in possession was exponentially larger due to PCR amplification. The only step remaining was to isolate the RNA aptamers from the DNA template and other artifacts. In order to do this, Polyacrylamide Gel Electrophoresis (PAGE) was performed. First the transcription product is submitted to DNase I treatment, in order to digest the DNA templates in solution. The sample was exposed to high temperatures and a denaturing dye to get rid of any RNA secondary structures. As shown in Figure 3, this method separates RNA strands into defined bands, according to their size. This way, the true aptamers are retrieved while the undesired artifacts are left behind.
Figure 3. PAGE gel electrophoresis showed a defined band of RNA aptamers.
After the RNA band was cut out, the gel was crushed and a solution containing NaOAc and TE buffer was added. This step takes the RNA strands out of the gel and into solution. The solution was then placed in a new tube to perform ethanol precipitation and Nanodrop spectrophotometry afterward. The Nanodrop analysis revealed the presence of RNA aptamers at a concentration of 1097.1 ng/uL. The fact that the concentration is relatively high demonstrates the level of success of this round of selection.
I came upon several problems early on in the selection process. Firstly, as I was readying myself to begin the round of selection, I realized that there was no more myostatin available in the lab. As a consequence, I had to wait 4 days until the new protein arrived. When the protein got here, I realized that it was not functionalized. This meant that I had to biotinylate the protein in order to follow through with the bead-based selection method. However, as stated previously, the total number of picomoles contained in my protein sample (400pmol) was so small that the biotinylation process might have been compromised. For this reason, I opted to switch to the filter-based selection method. The fact that myostatin is such a large protein further favored this decision, as filter-based selection is generally successful with large molecules only.
Another problem I encountered was my negligent lack of NTC during cycle course PCR. Without the NTC, I couldn’t tell if the band obtained corresponded to the aptamer or to DNA contamination. Therefore, I ran my lsPCR products, along with a NTC, through a gel. The NTC sample showed no amplification, which meant that the amplification present in the cycle course gel truly belonged to the desired aptamer dsDNA.
Conclusion and Future Work
As a final point, the first round of N58 aptamer selection against myostatin was successfully performed. Even though I came upon a few setbacks in the process, in the end the results obtained were satisfying. In the future, I will keep performing rounds of selection in order to increase the affinity and specificity of the aptamer. Moreover, I will collect the washes used during filter selection for the next rounds, as this time around I didn't do so.
I taught Elena how to perform filter-based selection
To see my abstract, click here.