I’ve always like the idea of aptamers – as generally used, that word refers to oligonucleotides that are selected for binding to something else (a protein target, for example). You get to use all the tools of molecular biology, which means that you can start out from insanely huge numbers of possible binders and select your way down (a bit like using DNA-encoded small molecule libraries, and feasible for the same kinds of reasons). The process is usually referred to as SELEX (systematic evolution of ligands by exponential enrichment), and it consists of solid-phase binding experiments to enrich for potential candidates, amplification of those, and further more stringent rounds of selection binding. If you’re using RNA aptamers, there’s a reverse-transcriptase step to send you into DNA space for the amplification by PCR; DNA aptamers can just roll right along. You can incorporate some mutation in the protocol if you like, and overall the number of variations on the SELEX idea would at this point be hard to count.
It can work dramatically well, if you’re careful to watch out for tight but nonspecific binders and so on. The resulting aptamers are useful reagents, although it’s generally hard to imagine them as drugs, being nucleic acids. If you could come up with equivalents for reverse transcriptase, PCR (high and low fidelity), and the rest for rounds of selection for more conventional small molecules, you would be in business for sure, and you’d put the rest of us out of business. DNA-encoded library screening is about the closest we’ve gotten.
The SELEX process, though, can be a lot of painstaking work, and from what I know about it, the majority of the time you don’t actually zoom in on any binders. One problem that’s been suggested is that the PCR step is actually a bit less likely to enrich the binding sequences, which may be more structured than the nonbinders, and there are influences from the composition of the starting library as well. But this new paper appears to be a rather startling improvement. It proposes “ideal filter” enrichment by capillary electrophoresis. In a standard CE experiment, just like with any other column or gel, you enrich by having one population move more quickly than another down the length of the device. The nonbinders wash off, for example, leaving the binders behind – but that’s a relative process, and if you have a bazillion nonbinders in the background with varying mobilities, things can get messy and smeared-out. This new technique actually makes the binders and nonbinders move in opposite directions, though. Which is quite a picture to contemplate, for those of us who are used to standard chromatographic column thinking.
The key is to increase the ionic strength of the running buffer. You’re tuning the mobility of the binders and nonbinders to the electroosmotic flow, and when you hit the right balance, you can get the ideal-filter effect. You also reduce the amount of binding to the sides of the capillary tube, which is one of those nonglamorous issues that can actually complicate assay technology a great deal. The authors showed this with a DNA repair protein called MutS, for which an aptamer (found by three rounds of traditional SELEX) was already available. Experiments with that one versus controls were very promising, so they tried a selection from a random-sequence library. One round of the high-salt conditions pulled out a group of aptamers with double-digit nanomolar affinities, while everything else went away. The signal/noise improvement was dramatic, roughly ten-million-fold better partitioning than the normal conditions.
So that sounds pretty useful, and as the authors point out, you might well be able to use these conditions for any other system where you have DNA attached to things – such as DNA-encoded library screening itself. I hope that this turns out to be as general a technique as it would seem!