Juan Herrejon
CCR5
N34 RNA Pool
October 18, 2011
Aptamer Stream Fall 2011
Progress Report 1
Progress, Results, and Discussion
After completing two successful practice rounds following the SELEX protocol described in the proposal of this research paper, the biotinylation of the CCR5 peptide was performed. The biotinylation protocol was followed using 1X HEPES buffer, the buffer in which the CCR5 was resuspened when it shipped. Using the Sulfo-NHS-SS-Biotin kit, the peptide was allowed to react with the biotin for two hours. The reaction was then purified and collected using Zeba Spin Columns; two subsequent washes using 1X HEPES buffer were also collected. These three samples were analyzed and their concentrations were recorded. At this point in the biotinylation protocol, an unusual phenomenon was observed in the concentrations of the three collected samples. The concentration of the reaction sample was found to be higher than expected, and the concentrations of the wash samples were abnormally high. In fact, the samples showed a higher concentration of protein than the sample that actually should have contained the biotinylated CCR5. Because of these results, Round 0 of the selection against CCR5 was delayed as the source of the problem is still unknown. Potential causes have been identified, such as the possibility of the spin column adding a specimen to the volume, and work is underway to solve the issue.
Once the issue has been resolved, the first round of selection against CCR5 will begin full force. The table below shows the parameters that will be followed in the first and subsequent rounds. The table does not show constant factors, such as pool (N34), buffer (10X HEPES), and beads (streptavidin).
Table 1: Summary of critical parameters and conditions for first rounds of selection.
Incubation Time | Incubation Temp | Wash Volume/No | RNA used | |
Round 0 | 45 mins | 37C | 3/1 | 400pmol |
Round 1 | 30 mins | 37C | 3/1 | 400pmol |
Round 2 | 30 mins | 37C | 3/2 | 400pmol |
Round 3 | 25 mins | 37C | 3/2 | 200pmol |
Round 4 | 15 mins | 37C | 4/2 | 200pmol |
Problems Encountered
Thus far, both the practice rounds and the actual rounds of selection against CCR5 have been problematic. During the practice rounds, the pellet for one of the washes (W0) flew out of the tube as it was being dried by the speedvac. Trusting that some of it must have stayed behind, the round was completed with successful results. For the second round, the gel electrophoresis for the cycle course PCR reactions showed up completely blank under UV light; not even the ladder showed up. The problem was identified in the ethidium bromide, as it worked after using a different aliquot. Despite these and other small problems, the practice rounds were successfully completed.
The first round in the real selection, however, has not been started due to a major problem encountered in the biotinylation of the CCR5 peptide (described in the progress, results, and discussion section of this progress report). Instead of showing negligible to no concentrations, the wash samples showed higher concentrations of protein than the sample that was supposed to contain all of the protein. The source of the problem has not yet been identified, as the many possibilities need to be weeded out one at a time.
Conclusion and Future Work
Thus far, several problems have arisen that have delayed the start of the first round of selection against CCR5. While the problems in the practice rounds were identified and successfully overcome, the problem encountered during the biotinylation of the target have not yet been resolved. Once the source of the issue has been identified, the selection can begin using the parameters identified in previous sections. Once Round 0 is completed, the resulting RNA will be utilized in the next round to decrease the amount of variants identified as potential aptamers. Subsequent rounds will weed out the weak variants under more stringent conditions to isolate the strands that exhibit the highest binding affinity to fibrinogen to make an efficient aptamer. In addition, all gels will be run with a no template negative control and negative selections will be performed every other round.
The link to my proposal can be found here here and my abstract can be found here.
This is the link to my Final Manuscript.
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