(Cell-)SELEX

To generate the aptamers for our test, we are using the Systematic Evolution of Ligands by EXponential Enrichment (SELEX) technique.

Aptamers are short single-stranded oligonucleotide molecules that bind specifically to a variety of target molecules1. These target molecules can be anything, from small organic molecules over proteins up to whole viruses and cells1. The high specificity and affinity of the aptamers to the target molecule are caused by the folding of the aptamers into a unique 3D structure that interacts specifically with the target1. We are using aptamers for our test instead of traditional antibodies, because aptamer sequences can be evolved quicker than antibodies and for a wider range of targets2. They are also cheaper and more versatile in production as they can be easily produced in vitro. Additionally, in contrast to antibodies, they do not require animals for production2.

The Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is a technique in molecular biology to evolve aptamers2. The procedure is based on the random specific binding and washing of non-specific aptamers to and from the target molecule and their subsequent amplification1.
There is a variety of SELEX methods available for various targets3. We used the Cell-SELEX technique, because we wanted to evolve aptamers that bind specifically to the cell surface of the plant pathogen Pseudomonas syringae which we used as a proof of concept for the detection.

Since Cell-SELEX is a difficult method, we invested a lot of time and effort to establish it in our lab. There were a lot of things we had to consider before we could even start with the different parts of our individual Cell-SELEX protocol. Needless to say, we had a lot of troubleshooting to do, once we actually started. To help future iGEM-teams that will use the Cell-SELEX method, we provide a detailed Cell-SELEX description. This will hopefully lead to a more efficient workflow.

1. Lab book SELEX | FILESIZE: 21MB
2. Lab book Cell-SELEX | FILESIZE: 3MB

Figure 1: Schematic representation of aptamer evolution through Cell-SELEX.

Target cells

In the Cell-SELEX method, the aptamers bind to molecules on the extracellular surface1. Cell-SELEX uses live cells and reducing the amount of dead cells in the solution significantly enhances the enrichment of the potential target aptamers1. Our targets were the cells of the plant pathogen Pseudomonas syringae. We calculated the amount of cells needed for each SELEX-cycle using the OD of an overnight culture. We then made a cryo culture and aliquoted it, so the culture wouldn't have to go through multiple freeze-thaw-cycles, which can be harmful for the cells. Before using a cell-aliquot in the cycle, we centrifuged and washed the cell suspension two times to receive a clean cell pellet. The supernatant was removed and the pellet was resuspended in a binding buffer. The binding buffer consists of DPBS, BSA and Tween 201, 2.

Library

The library contains about 1014 different oligonucleotides, which share a randomized region of 30-50 nucleotides2. The randomized region is flanked by two conserved regions2. These conserved regions function as primer binding sequences in the amplification step. The regions we used were designed by Prof. Dr Günter Mayer and supposedly reduce the amount of miss amplification4. Because of the sheer number of sequences it is likely that a few of them are able to bind to the target with high specificity. Before use, the library was mixed with the binding buffer, heated to 95°C for 10 minutes to break the nucleotides down into their primary structure. They were then snap-cooled on ice to keep this structure. This is essential for the aptamers to exhibit their maximal binding potential.

Positive selection

After mixing the library with the target cells, some of the oligonucleotides will bind to the target with varying binding affinities. To increase the specificity of the aptamers, you could also combine the positive selection with a subsequent negative selection. This method is useful to eliminate nontarget cell-specific oligonucleotides5. This is possible due to the binding of the aptamers to the specific molecules on the extracellular surface of the cells1. So the negative selection filters out sequences that may bind to molecules existing on the surface of both the target and the different cell lines1. After some negative selections most of the nonspecific bound aptamers can be eliminated and they will not be amplified in the selected pool1.
After we mixed the target cells and the library together, we incubated the mix on a shaker at 500rpm for 1h at 4°C.

Extracted bound sequences

Now sequences that are bound to our target cells and the unbound sequences need to be seperated. To remove the aptamers that did not bind to our cells, we used Amicon Filters (0.5 ml 100 kDa cut off). The Amicon Filters let the unbound aptamers flow through and the aptamers bound to the target cells remain in the residue, as the cells cannot pass through the filter. The target cells with the bound sequences then get heat shocked at 95°C, to denature the proteins on the cell surface and to revert the aptamers binding1. After the heat shock, the mix has to be centrifuged again, to separate the aptamers from the cells.
After a couple of centrifugation and washing steps, we heat shocked the cells to destroy them. Through centrifugation, we created a pellet again, to remove all the cell debris. The aptamers are located in the supernatant and can now be collected.

PCR amplification

To amplify the extracted sequences, we used PCR amplification. The determination of the optimum cycles is very important, because too many cycles can lead to nonspecific amplicons1.
The primers we used for our Cell-SELEX were also designed by Prof. Dr. Günter Mayer4. We used his primers to reduce misamplification during the PCR. The primers were also 5'-phosphorylated, to create single stranded DNA in the next step. First we did a preparative PCR with 8 cycles to increase the amount of sequences in general. After that we used 5 different samples to determine the right amount of cycles. We stopped the reactions after 4,6,8,10 and 12 cycles. We used a gel electrophoresis to determine the right amount of cycles, but as there were no visible bands, we could not determine the right amount. Because of the lack of time we were not able to establish a finished PCR procedure for our cycle.

Figure 2: Electrophoresis to determine the right amount of PCR cycles

Gel:

3 % TAE Agarose

5 µl Gel red in 50 ml

Lane (from left to right):

1. 100 bp ladder
2. ssDNA pool
3. After 8 rounds of PCR
4. After 8 rounds of PCR + 4 rounds of PCR
5. After 8 rounds of PCR + 6 rounds of PCR
6. After 8 rounds of PCR + 8 rounds of PCR
7. After 8 rounds of PCR + 10 rounds of PCR
8. After 8 rounds of PCR + 12 rounds of PCR

Digestion

After the PCR reaction the oligonucleotides are now double stranded. But the aptamers need to be single stranded DNA. For the next cycle, the 5'-phosphorylated strands are digested by a lambda exonuclease. In our case we used the lambda exonuclease from New England Biolabs (catalog #M0262S). The Lambda Exonuclease is a DNA-specific exonuclease. It is a highly processive 5'-3' exonuclease that selectively degrades 5'-phosphorylated strands6. The exonuclease prefers 5'-phosphorylated strands, due to the formation of inert enzyme-substrate complexes7. Since we had to prove that the lambda exonuclease works as intended for our cycle, we worked with different methods. First we tried to prove it by measuring the concentration of single stranded DNA with the Nanodrop by ThermoScientific. But a better and more visual verification was done with a 2 % TBE Agarose gel. The digested samples (single stranded DNA) traveled a longer observable distance through the gel compared to the undigested samples (double stranded DNA).This indicates the successful digestion of our sample.

Figure 3: Electrophoresis for digestion verification.

Gel:

2 % TBE Agarose

2,5µl Gel red

75V for 90 minutes

Lane (from left to right):

1. ssDNA (Clean up after digestion)
2. dsDNA Reference (Probe 1)
3. ssDNA (no clean up after digestion)
4. 50 bp ladder

synthesised aptamers

The whole process can now be repeated. After a variable amount of positive SELEX-cycles (typically 6-20 cycles), aptamers with a high binding affinity and specificity can be evolved for the cells of our target, Pseudomonas syringae.

iGEM wikisync tool

Thanks to last year's iGEM Team BITS Goa and their very useful tool called igem-wikisync we could head start our wiki and have a well thought out workflow. Unfortunately for us (and we are sure for some other teams too) it had some minor flaws regarding UTF-8 encoding and special characters.

References

1. Sefah, K., Shangguan, D., Xiong, X., O-Donoghue, M. B., & Tan, W. (2010).

   Development of DNA aptamers using Cell-SELEX.

   Nature Protocols 5(6), 1169-1185.

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2. Mayer, G. (2016).

   Nucleic Acid Aptamers.

   Springer New York.

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3. Gopinath, S. C. B. (2006).

   Methods developed for SELEX.

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4. Mayer, G., Ahmed, M.-S. L., Dolf, A., Endl, E., Knolle, P. A., & Famulok, M. (2010).

   Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures.

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   BioResearch Open Access 1(6), 265-272.

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6. Avci-Adali, M., Paul, A., Wilhelm, N., Ziemer, G., & Wendel, H. P. (2009).

   Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation.

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7. Subramanian, K. (2003).

   The enzymatic basis of processivity in lambda exonuclease.

   Nucleic Acids Research 31(6), 1585-1596.

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