In this paper, the authors use a new method called micro free flow electrophoresis to find aptamers against IgE. This device works by separating bound aptamers from unbound aptmers by using an electrical charge, where bound aptamers should be less anionic and migrate differently. The figure below shows the best binds are found after the first round of selection without the need to do a negative selection. The authors also found the dilution to be only 100 fold which is 60 fold less than what is seen in a SELEX experiment.
I find it a bit strange that the enrichment of the DNA pool is not driven by selective pressure. The power of SELEX is the ability to enrich binding affinity by applying pressure within the selection parameters. It seems that their method for separation (charge relationships) does not further enrich after one round, which would indicate there is a limitation within their system and that the "evolution" phase of the SELEX is very weakly modulated. This could work as a method to quickly develop DNA SM binders, but is limited in the ability to drive enrichment beyond that first round. Would have been good to run a "standard" SELEX with bead capture of the IgE to compare the enrichment over rounds.
ReplyDeleteNicole, great find! I hadn't though about electrophoresis as a tool for aptamer selection before.
ReplyDeleteJames, you raise an interesting point, but I'm not sure it's safe to say that their selection is weaker than SELEX. Their results indicate that their aptamers perform nearly as well as those found by SELEX (30-50 nM Kd vs 10 nM from SELEX) after just a single round. It's possible that the technique results in so few false positives that it yields the same result as 10+ rounds of SELEX in a single round.
I'm interested in other forms of electrophoretic aptamer selections. You could imagine coupling a molecule of interest to acrylamide and adding that into a highly crosslinked SDS-PAGE resolving gel mix. That way you end up with your crosslinked polyacrylamide matrix, but with your molecule of interest covalently attached. Run your aptamers through the gel. Aptamers that don't interact with the ligand will flow through very fast. Those that do will run more slowly depending on how tightly they bind to the ligand and, unfortunately, how greatly their effective charge is diminished. To remove aptamers that bind to the matrix but not the ligand, you could do a negative control without your modified acrylamide and pull out the fastest aptamers. You could increase selection strength by increasing the current...
Idk, I'm sure someone's tried this.
Indeed, this methodology is interesting. Perhaps the size of aptamer libraries searched through here (10^14) are slightly larger than those from SELEX? In addition, this approach is done in solution, so specificity towards a solid support is evidently not an issue - unlike with traditional SELEX, whereby a large fraction of aptamers non-specifically bind the support. That may be why further rounds of enrichment didn't improve the binders. I reckon its a bonus that such good binders were found after just the one round! In fact, these particular aptamers compared very will to those identified by SELEX.In addition, I suspect the range of allowable selection conditions for this approach is better than that offered by resin-based SELEX.
ReplyDeleteYes, it seems like using this method, you get the tightest binders available in your library after one round. It would have been interesting if they put in the sequence obtained from SELEX, and seen if that same sequence was obtained by this method after only one round. Johnny, I am not sure if you can use an acrylamide gel. Putting these aptamers in a gel, you will not only separate by charge but also by size, and since most library members will have a different 3 dimensional structure, they will all probably run different on a gel. The reason I liked this paper so much, in addition to finding the tightest binders after only one round, was because it eliminated the negative selection step. I believe this method now opens the door to continuous directed evolution of aptamers.
ReplyDeleteRemember that they're finding an aptamer that binds a protein. The effective charge of the aptamers might not change so dramatically upon binding a small molecule. We don't know how well this would work for small molecules.
ReplyDeleteNicole, separation because of the differences in 3D structure would be very, very minimal. If the % crosslinking is chosen well, sequences that don't interact with the ligand would just fly through the gel, regardless of how they fold. Also, how could this be used in a continuous fashion? I'm definitely not seeing it. It's repeatable, but that's not the same thing.
Yeah, your right Johnny, I imagine if you do use a gel, the aptamers that don't bind will flow quite quickly, but then how do you visualize and remove your aptamers from the gel without loosing yield?
ReplyDeleteAs far as the continuous directed evolution, I am not sure of the mechanics because I am not an engineer. However,I can imagine using this method and getting the best binders after only one round. Then directing the flow of the aptamers that do bind into a round of mutagenic PCR using some kind of microfluidic devise. I also believe that could also increase the flow over time so that you can add selective pressure, removing those that don't bind as quickly. Would this not be continuous evolution?