Angiostatin Selection Progress Report

Tianlu Ma

October 17, 2011

Fall 2011

R50, RNA, Angiostatin

Progress Report 1

Progress, Results, and Discussion

Since the recombinant human angiostatin protein (K1-3) was approximately 30 kDa, filter based selection was performed. In preparation for selection, the dried protein stock was suspended in 33.3 μL of 0.1 M HEPES buffer, pH 7.5, giving a final 100 μM concentration. Since approximately 200 pmol of protein will be needed per round, the stock solution was aliquoted out in 2 μL volumes. Additionally, 40 mL of 1X PBS buffer was prepared (diluted from 10X stock). A 1 M Tris-HCl solution, pH 7.37, was prepared, and this solution was used to make 40 mL of 10X Tris Selection Buffer, containing 200 mM of Tris-HCl, 1370 mM of NaCl, 50 mM of KCl, 20 mM of CaCl2, and 30 mM of MgCl2. While the buffer preparation itself was not hard, the calculations did take a while due to the fact that I haven’t made buffers in a while.

The selection itself began with a binding reaction. Pool R50 RNA was selected for selection, and the pool binding reaction was made with approximately 400 pmol of the R0 RNA and 1X Tris Selection Buffer. This mixture was heated to 65°C for RNA denaturing and allow the RNA to unfold. The 300 pmol of protein was then added and the final mixture was incubated at 37°C for 30 minutes. The nitrocellulose filter was set up and washed 3 times using 1X Tris selection buffer. The incubated protein-RNA mixture was then added to the filter and, after having pushed through the liquid so that only the RNA that was bound to the protein stuck to the filter, the filter was taken out and placed into a 0.6 mL tube, with the side exposed to the protein-RNA mixture facing the inside of the tube. It was noted that it appeared as though the filter had an “edge” that had started to come off, however there was no actual tear on or near the center of the filter. The RNA was eluted off the filter using 2 washes of 200 μL Elution Buffer (stock). Each wash was heated at ~90°C for 5 minutes to facilitate the elution process, then placed on ice for a minute to prevent aerosols. The 2 washes were combined into one 400 μL volume and ethanol precipitation was performed to concentrate the RNA. The pellet was resuspended in 10 μL of diH2O. Reverse transcription was performed with 8.5 μL of the resuspended RNA, 20 μM of R50 reverse primer, and 500 μM of dNTPs. This was heat denatured for 5 minutes at 65°C, and 1X First Strand Buffer, 10 mM DTT, and 1 μL of SuperScript II RT enzyme was added. The final solution was placed in a thermocycler, which ran the reverse transcription program (42°C for 50 minutes, 70°C for 15 minutes).

Amplification of the pool was done with PCR. Cycle course PCR was performed using 1X PCR buffer (lab stock), 200 μM dNTP, 400 nM R50 T7 forward primer, 400 nM of R50 reverse primer, 2 μL of the ssDNA from reverse transcription, and 2.5 U of Taq DNA polymerase. During cycle course, when a sample for cycle 10 was ready to be obtained, the lid of the thermocycler used broke, resulting in a few minutes delay from cycle 10 to cycle 11. Fortunately, the DNA appears to have amplified correctly, and, from the gel in Figure 1, it can be seen that 10 cycles was optimal. Therefore, 10 cycles were run for large scale PCR with 1X PCR buffer (lab stock), 200 μM dNTP, 500 nM R50 T7 forward primer, 500 nM R50 reverse primer, 2 μL of the ssDNA obtained from reverse transcription, and 2.5 U of Taq DNA polymerase. Ethanol precipitation concentrated the PCR product for transcription, and the product was resuspended in 20 μL of diH2O. A transcription reaction was prepared using 1X Ampliscribe Transcription Buffer, 10 mM DTT, 7.5 mM of each NTP, 5 μL of dsDNA from lsPCR, and 2 μL of T7 enzyme solution. The reaction was incubated at 42°C for approximately 6 hours.

Figure 1. R0 R50 Angiostatin Selection cycle course PCR. The gel shows that 10 cycles is the optimal number of cycles for this round of PCR. NTC shows some signs of primer amplification, but thankfully does not show an actual band.

Problems Encountered
As stated above, the broken thermocycler was a big issue for this round. However, since the thermocycler has been fixed, hopefully another won't break right when I'm using it. The only other problem would be the fact that a single transcription kit will rarely have all the needed components, leading to an increase in the chance of a possible contaminate reaching the selection process.

Conclusion and Future Work
In the upcoming week, a PAGE gel will be run to determine if any RNA product was obtained. If a product was obtained, it will be quantified using nanodrop spectrophotometry. Results condensed in Table 1. Since it seems that filter based selection takes much less time than bead based selection, I am hopeful that I can get at least 1 round done per week. To prepare for assaying, I will start looking into radiation handling classes. If or when I obtain an aptamer by selection with Tris buffer, I will then obtain MES buffer and select for angiostatin under the conditions of the tumor microenvironment.

Round	PCR Cycles	Peak Absorbance (nm)	Initial RNA Amount (pmol)	RNA Yield (pmol)
1	10	260	~400	1561.20
2	N/A	N/A	~400	N/A
3	N/A	N/A	N/A	N/A
4	N/A	N/A	N/A	N/A
5	N/A	N/A	N/A	N/A

Table 1. For R50 RNA Selection of Angiostatin K1-3. This table shows the number of cycles run for PCR, the absorbance, the quantity of RNA used, and the quantity of RNA recovered. Added October 21, 2011. Last edited October 30, 2011.

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