The overall goal of the following experiments is to generate RNA Aptima in vitro for HIV one GP one 20 protein using a nitrocellulose membrane. This is achieved by PCR and subsequent in vitro T seven transcription. To prepare the initial DNA and random RNA library as a second step, the mixture of the target protein and RNA library is applied onto a pre wetted nitrocellulose filter, which allows isolation of bound RNA molecules from unbound RNA species.
After 12 selection cycles, the RNA library is cloned and sequenced in order to identify the individual RNA aptima candidates from the library. Results are obtained that identify several new aptima with nanomolar affinity based on gel shift assays and confocal microscopy. The main advantage of this technique over existing measures like bets or column based isolation measures is simple and doesn't required a pretreatment of the protein or libraries such as the immobilize of the protein on the bes, The RNA library for the isolation of anti HIV GP one 20 RNA Aptus is prepared from a commercially synthesized DNA library that contains 50 nucleotides of random sequences.
The single stranded DNA oligo library sequence is 81 nucleotides in length. As shown in this diagram, the random region is flanked by constant regions, A 30 tumor at the five prime end, and a 16 MA at the three prime end. The constant regions include the T seven promoter for in vitro transcription, and a three prime tag for R-T-P-C-R.
Amplify the single stranded DNA oligo random library by PCR limit PCR to 10 cycles in order to preserve the abundance of the original DNA library. After the PCR reactions, the amplified double stranded DNA pool is recovered using a gel purification kit. The double stranded DNA is then converted to an RNA library using the cript kit according to the manufacturer's instructions in the transcription reaction mixture replaced CTP and UTP with two fluoro pyrimidine, CTP and two fluoro pyrimidine UTP to produce ribonuclease resistant RNA.
When the transcription reactions are complete, digest the reactions with DS one to remove template DNA and purify by an 8%poly acrylamide, seven molar urea gel. Finally, quantify the purified RNA library by UV spectra phyto. Prior to the selection of optimas, prepare the required buffers and store them at the appropriate temperatures.
Heaps buffer at room temperature and buffers for RNA refolding and RNA binding of minus 20 degrees Celsius. Before starting the first cycle of selection, preor the random RNA pool to a pre wetted nitrocellulose filter at room temperature for 30 minutes to minimize nonspecific binding with the filter. After 30 minutes, remove the nitrocellulose filter.
Combine the pre-cleared random RNA pool and HIV one bowel GP one 20 protein in low salt RNA binding buffer incubate on a rotating platform at room temperature for 30 minutes. Next, pass the reaction through a pre wetted nitrocellulose filter and wash with one milliliter of low salt RNA binding buffer to elute the bound RNA from the filter. Remove the nitrocellulose membrane from the filter holder.
Add 200 microliters of elu buffer to the membrane and heat at 95 degrees Celsius for five minutes. After the RNA has been eluted from the filter, spin the tube briefly transfer the RNA solution to a new tube and perform a phenol chloroform extraction on the eit. Use a MicroCon YM 30 column to concentrate the RNA and elute the recovered RNA with 20 microliters of RNAs free water.
The recovered RNA pool is then reversed, transcribed, and amplified for 15 cycles of PCR. After the amplified double stranded pool is purified, it is transcribed into a new RNA pool as demonstrated previously before the next selection round. Refold the RNA pool in one times HBS buffer heat to 95 degrees Celsius for three minutes, and then let slowly call to 37 degrees Celsius.
Continue incubation at 37 degrees Celsius for 10 minutes. Adsorb the refolded RNA, pull to a nitrocellulose filter for 30 minutes prior to incubation with the target HIV bowel GP one 20 protein for the next selection round. In order to successfully obtain the abers with the higher bind infinity, the selection stringency must be carefully controlled by adjusting selection conditions.
The selection conditions are shown in this table. For cellex rounds one to four, incubate the pre-cleared RNA pool with the target protein in low salt RNA binding buffer for 30 minutes. After the fourth round of cellex, use high salt RNA binding buffer as cellex progresses.
Reduce the amount of GP one 20 protein and increase the competitor yeast TRNA in order to increase the stringency of aptus selection at each cycle. The progress of HIV one, GP one 20 optima selection is monitored by a filter binding assay. In this video, the 12 RNA pools collected after all the selection cycles will be assayed.
First, remove the initiating five prime tri phosphate by treating each RNA pool with calf intestinal phosphatase for one hour at 37 degrees Celsius. To prepare the CIP treated RNA for labeling with T four polynucleotide kinase and P 32 80 p heat at 95 degrees Celsius for five minutes, followed by chilling on ice labeling of the RNA is performed in a hot room designed for experiments involving radio activity at PNK buffer, T four Polynucleotide kinase, one microliter of gamma P 32 A TP and water to 20 microliters. Incubate at 37 degrees Celsius for 30 minutes.
After 30 minutes, add 20 microliters of water to each reaction and purify by a G 50 column. Finally, obtain 40 microliters of labeled RNA as a final concentration of 250 nano molar refold, each labeled RNA pool in one times HBS buffer heat to 95 degrees Celsius for three minutes. Following this, allow to call to 37 degrees Celsius, which will take 30 to 40 minutes.
Continue incubation at 37 degrees Celsius for 10 minutes. When the RNA pool has been refolded, carry out a 100 microliter binding reaction. Combine the end labeled RNA pool with GP one 20 protein and a tenfold molar excess of non-specific competitor TRNA in high salt RNA binding buffer incubated room temperature for 30 minutes.
At the end of the 30 minutes, separate 50 microliters of the binding reaction by a pre-wet nitrocellulose filter. Then wash the filter with two milliliters of binding buffer count the radio activity retained on the filter using a multipurpose scintillation counter as an input control count. The remaining 50 microliters of binding reaction at the same time, the binding assay of the RNA pool after each select cycle was analyzed by a filter binding assay with competitor TRNA to monitor the progress of HIV one, GP one 20.
Optima's selection binding activities were calculated as the percentage of input RNA retained on the filter in protein RNA complex as shown here. The starting RNA pool only shows 0.1%of the input RNA retained on the membrane. However, after nine selection rounds, the ninth RNA library has 9.72%of the input RNA bound.
Although additional selection rounds were conducted, no further enrichment is observed suggesting that maximal binding of the RNA pool has been reached. The binding activities of the RNA pools were further confirmed by gel shift assays. These results demonstrate that some ligands with high binding specificity for the target protein are successively enriched in these RNA pools.
In this example, the 12th RNA pool showed the highest binding activity since maximal binding of the RNA pool was reached following the 12th round of selection, this RNA pool was reversed transcribed to CDNA, which was subsequently amplified by PCR. The PCR product was then purified and cloned into a TA cloning vector and sequenced after alignment of 140 clones. Six groups of anti GP one 20 optimas were identified.
Only the random sequences of the Aptima core regions are indicated in this table. About 40%of the clones contain a conserved sequence. One representative sequence from each group was chosen for further characterization because of their relative abundance within their group through a native gel mobility shift assay, the dissociation Constance KD of these representative abers were calculated, for example, A one.
The best of the abers has an apparent KD value of 52 nanomolar. As shown here. These selected abers can selectively bind with the target HIV one bowel GP one 20, but not the H-I-V-G-P one 20 CM protein CHO G one 60 cells stably expressing the HIV envelope glycoprotein GP one 60 were used to test for binding and internalization of the selected anti GP one 20 optimas.
These cells do not process GP one 60 into GP one 20 and G 41 since they lack the gag encoded proteases required for envelope processing flow cytometric analysis reveal that the S3 labeled abers specifically bin into the CHO GP one 60 cells, but not the control cho EE cells, which do not express GP one 60. Furthermore, real time live cells Z axis confocal microscopy indicates that after two hours of incubation, the SI three labeled aptima is selectively internalized by the CHO GP one 60 cells, but not by the cho EE control cells. It was also observed that the aptima aggregated within the cytoplasm, suggesting that the GP one 20 aptima may enter cells via receptor mediated ptosis.
Finally, after you watching this radio, you should have a good understanding of use, how to use the field based measures to isolate RNA Abers.
