Current Progress on Aptamer Selection for gag p24

The abstract to this proposal can be found here

Progress, Results, and Discussion

Three rounds of selection using N34 RNA pool, gag p24 as the protein target and PBS SELEX as the buffer solution have been performed.

Perameters for the First Round of Selection:

RNA:Protein- 400pmol:200pmol

Negative Selection- 40ul for 50 minutes

Selection with Protein- 25 minutes

Wash Volume of 1X PBS- 200 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Perameters for the Second Round of Selection:

RNA:Protein- 400pmol:200pmol

Negative Selection- 40ul for 32 minutes

Selection with Protein- 23 minutes

Wash Volume of 1X PBS- 200 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Parameters for Thrid Round of Selection:

RNA:Protein- 200pmol:100pmol

Negative Selection- 40cl for 32 minutes

Selection with Protein- 15 miuntes

Wash Volume of 1X PBS- 300 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

There are two remarkable results that can be seen in my first and second cycle course. The first is that despite a lengthy negative selection in the first round, my elution visibly amplified at twelve cycles. Also, in both my first and second rounds, there is no visible amplification in the W3 and a good amount of amplification in the elution, despite the low wash volumes and the early stage of the selection. From the first to second round of selection, I didn't want to change my conditions too much, because I wanted to allow my pool to stabilize. However, when I received the same promising results in the second cycle course as I did in the first cycle course I decided to to increase the stringency of selection. This led to a large amount of RNA in both washes and the elution, which is at first glance odd, because I increased my wash volume. However, I postulate that the increase of RNA in my third wash is due to the decrease in incubation time and possibly due to the decrease in protein. Next round, I plan to perform a +/- selection to see if the large amount of RNA in my elution is due to protein binding or bead binding. I am also going to drastically increase my wash volume to 500ul per wash.

Three more rounds of selection using N34 RNA pool, gag p24 as the protein target and PBS SELEX as the buffer solution have been performed, since the last update.

Parameters for the Fourth Round of Selection (+/-):

RNA:Protein- 200pmol:100pmol

Negative Selection- 40ul for 33 minutes

Selection with Protein- 15 minutes

Wash Volume of 1X PBS- 500 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Parameters for the Fifth Round of Selection:

RNA:Protein- 200pmol:100pmol

Negative Selection- 80ul for 39 minutes

Selection with Protein- 12 minutes

Wash Volume of 1X PBS- 500 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Parameters for the Sixth Round of Selection:

RNA:Protein- 150pmol:100pmol

Negative Selection- 80ul for 38 minutes

Selection with Protein- 10 minutes

Wash Volume of 1X PBS- 500 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

The most important thing to note about these cycle courses is that in the R4 +/- selection the E4(-) amplifies earlier than the E4(+), so there may be substantial nonspecific binding. A filter based binding assay was performed after the sixth round of selection, there was no relevant binding. The results of the binding assay can be seen below. It should however be noted that the binding assay was done on a filter, where as the selection was performed on magnetic beads. Also, tRNA was used in the binding assay and not in the selection. Both of these factors could contribute to the low amount of binding.