As the first round of selection has not been completed yet, this report will mostly focus on the decisions made about the different types of parameters and future plans for the selection process against alpha-Synuclein (a-syn). First of all, it was decided that filter selection would not be an option for selecting against the protein as a-syn is only 14.4 kDa in weight and would most likely be passed through the filter. Therefore, bead based selection is the best option for a moderately sized protein like a-syn. The aliquots of protein available in the -80C freezer were not tagged in any way, meaning that the protein was in need of being tagged with biotin. The biotinylation process required a lot of time in order to ensure that the protein was sufficiently tagged and could be effectively bound to the strepdavidin beads. The protein was originally stored in water in aliquots that had concentrations of 69uM.
Based on the Nanodrop Spectrophotroscopy there is a sufficient amount of protein remaining after the isolation of the target. This means that the first round can begin as soon as the protein is aliquoted into volumes that contain about 400pmol. There were some issues that were associated with the biotinylation of the protein, however. First off, the reagent was not immediately added to the protein as the protocol stated to do. The lag time between activation of the biotin reagent and the addition of the protein was around 30 minutes. The reason that the biotinylation was allowed to proceed though was the notion that there should be at least some of the biotin remaining that was unreacted and could still react with the a-syn. Secondly, more protein was added to the biotin reaction than was originally thought based on the labeling of the aliquots of the a-syn in the -80C. It has yet to be determined if this will affect the level of biotinylation of the protein and how to best remedy that situation without performing the biotinylation a second time. Another issue was that the biotin and a-syn complex was not purified from the rest of the reaction components until several days later because of time constraints.
There should not have been a problem with the biotin due to these two points of contention; however if a problem of protein binding should arise, these will be the events considered as possible causes. The concentrations of the remaining protein were calculated using Beer-Lambert’s Law. The extinction coefficient for a-syn was found by and online extinction coefficient calculator to be 5120 M-1cm-1. The concentrations of protein samples after the biotinylation process are listed in the table below:
The next part of the selection process that was completed was the creation of parameters and stringency for the rounds of selection. The parameters for the first five rounds of selection are listed in a table at the end of this report. The pool chosen for this project was N35, it was selected arbitrarily in an attempt to select a pool from the lab that had not developed amplicons. The criteria for the incubation of the RNA with the bead-protein aggregate is going to start at 25 min at 37C in order to ensure that binding occurs at body temperature as the aptamer has a downstream in vivo application; however the incubation time will decrease over the course if several round to increase affinity of possible aptamers. The buffer chosen for the binding reaction is Tris with salts at a pH of 7.3. This was chosen because the pH of cerebrospinal fluid is around 7.3 and mimicking the pH of the brain is crucial for the successful application of the aptamer. The washes will be done with 2 volumes of 200ul and there will be 3 washes done; although, this will gradually increase over the course of several rounds.
Also during the time before this first report I created three different buffers because they had run out. They have my name on them, but I made about 10mL of each so there should be plenty to share if someone needs them. I also did a lot of extra journal research in order to make sure that I knew every aspect of my target possible before I completed my round in order to ensure that the correct parameters had been chosen. There were several occasions whenever I came in to work on journal research and writing in my lab notebook that I helped some of my fellow Aptamer students think their way out of issues with their targets and selections. Although there is not a lot of data to be shown for this reporting period, it was a very productive couple of weeks – I learned a lot about a-syn and the selection process as a whole by taking the time to go slow before jumping into rounds of selection.
In conclusion, the preliminary steps the needed to be done before the selection rounds could begin have been thought of, outlined, and performed. The next steps that will occur will be to begin the first round of selection. I plan to complete about one round per week until the end of the semester. This means that by the next reporting period I should have completed around 3 rounds. This table indicates the aspects of the parameters of selection that are usually made variable over the course of several rounds of selection in order to increase affinity of the aptamers found. These parameters are likely to change, this is only a proposal of the possibly variations.