Touria Rguig
Tr7356- October18th, 2011
R50- Hemoglobin S
Progress, Results and Discussion:
Due to the large size of Hemoglobin S, filter based selection was performed instead of bead based to separate bound species from unbound species, the incubation time between the hemoglobin S and the RNA pool (R50) is very important, in that the bound and unbound RNA species could come to an equilibrium state; reducing the incubation time can lead us to select faster binding kinetics but in the same time it reduces the number of RNA sequences that get selected. Therefore, for round 0 we incubated 200 pmol of target and 400 pmol of pool R50 for 35 min at 37 C using Tris buffer. Tris buffer was an ideal choice for a selection buffer because it corresponds with the isoelectric point (7.09) of hemoglobin S. The eluted bound RNA species tend to have tight binders with slower off rate kinetics.
During the ethanol precipitation we eluted the RNA pool further so that we can only look at the very specific bound RNA sequence. After the RNA pool was concentrated we amplified the RNA pool and converted it to a single stranded DNA using reverse transcriptase. In this part, we run a cycle course PCR on the elution from the selection step to check for the optimal number of PCR cycles for large scale amplification. We only took a 5uL samples from the end of cycles 8, 10, 12, and 14 and 20. We then run those cycles in a 3.8% agarose gel at 100 V for 30 min to check out which cycles are optimal for large scale amplification for further experiments.
The gel in Figure 1 shows the amplification of the eluted species at different PCR cycles. E1 showed ideal amplification between cycles 8-10. So, we chose cycle 9 for the large scale PCR of E1.However, the “no template control” showed a band which can be due to many reasons such as contaminations in the primers or the pool itself. Another possibility could have been that the PCR cycle didn’t run properly due to a problem with the thermocycler. The large scale PCR of 9 cycles was going to be potentially used for transcription.
We started another round of selection using the same pool R50 but different aliquots of the primers, after selection and reverse transcription. We are now in the process of running another 20 cycle course PCR to choose the ideal number of cycles for the large scale PCR of the eluted bound RNA.
Problems Encountered:
There was only one problem encountered during Round 0 of the selection process. The no template negative control showed a band during cycle course PCR which creates a big problem. A band in the no template negative control could potentially be due to a contamination in the primers or some other species was transformed into the PCR tubes due to poor pipetting techniques. In order to troubleshoot the problem, another round of selection was repeated using different aliquots of primers and using proper sterile techniques when pipetting.
Bonus:
In addition to making aliquots of 100 bp DNA ladder, glycogen, and RNA pool. I also helped a few students with their PAGE gel and the filter based selection technique. Furthermore, I organized the lab a few times.
Conclusion and Future Work:
Aptamers are nucleic acids that bind tightly to a specific target, they are used in research, therapeutics, diagnostics and drug delivery. In this research we are selecting for an aptamer that blocks the Valine’s hydrophobic interactions with neighboring amino acids in the protein Hemoglobin S. This interaction causes blood cells to become sickled which prevent blood flow and transportation of essential nutrients and gas exchange. After getting a band in the no template negative control which was due to a contamination in the primers, this caused us to repeat the entire selection from round 0.
I am now starting a new round of selection, using the same pool (R50) but with different primers. I just finished reverse transcription. Next, I plan on carrying cycle course PCR, large scale PCR and amplify the transcribed DNA to RNA and then run it on an acrylamide gel in order to be able to quantitate the RNA product for assay. Figure 2 shows a prospective outlook on the way I plan on varying future rounds of selection.
Figure 2: Current and future progress on the RNA selection against Hemoglobin S.
| Round number | Quantity of protein/RNA used | Number of Washes/volume | Incubation time |
| 0 | 400 pmol/ 400 pmol | 3 washes /1 volume | 35 min |
| 1 | 400 pmol/ 400 pmol | 3washes/2 volumes | 35 min |
| 2 | 800 pmol/ 400 pmol | 2 washes/ 1 volume | 30 min |
| 3 | 800 pmol/ 400 pmol | 2 washes/ 2 volumes | 30 min |
Here is a link to my proposal and abstract: https://docs.google.com/document/d/1so6mZ7vjYMlsYqiFvoKlozjp6_6ktJcX73dZ-vkq9xM/edit?hl=en_US
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