In this work, we have demonstrated that M-SELEX provides a highly efficient and broadly applicable platform for the selection of high-affinity aptamers against a variety of protein targets. Using this method, we performed selections against three different proteins with pIs ranging from 9.3 to 5.3. Within three rounds of selection, we obtained a new aptamer sequence that binds to PDGF-BB with a Kd
of 0.028 nM, and to thrombin with a Kd
of 0.33 nM. These affinities are significantly higher than those previously reported in the literature 
, which also required considerably more rounds of selection to generate. We also report a new aptamer targeting ApoE with a Kd
of 3.1 nM.
The results obtained with these three targets suggest that the target protein charge state may exert some degree of influence on the overall affinity of the selected aptamer pool. To follow up on this finding, we performed aptamer selections for PDGF-BB at different buffer pHs using the same selection protocol which revealed an apparent inverse correlation between protein pI and aptamer Kd and therefore provided additional support for this initial observation.
To explore whether this relationship between a protein's charge state and aptamer affinity potentially represents part of a broader pattern, we plotted the Kd
s of 75 published aptamers (Table S3
) as a function of the pI of their target protein (). pI reflects the average net charge of a protein and does not describe the spatial charge distribution, but nevertheless offers a useful metric to make a general observation of a trend. In this scatter plot, the relationship between aptamer affinity and the protein's isoelectric point is not immediately obvious, presumably due to the differences in selection conditions and variability in affinity measurement methods.
Aptamer affinity versus isoelectric point.
However, when we examined only the subset of aptamers with Kd<1 nM, we observed a clear trend. For targets with pIs ranging from 4.0 to 6.0, only 11% (2/19) of the resulting aptamers exhibited subnanomolar affinity, but this percentage increased to 23% (3/13) for aptamers isolated against targets with pIs ranging from 6.0 to 8.0 and to 35% (15/43) for aptamers against proteins with a pI greater than 8.0. Based on this analysis, we observe additional evidence for a relationship between the overall net charge of a target protein and the affinity of aptamers selected against that target.
Electrostatic surface potential maps of the three protein targets selected here offer a potential explanation for this trend (Figure S5
); the protein with the highest proportion of positively-charged surface area (PDGF-BB) yielded the highest affinity aptamer, whereas the protein with the lowest proportion (ApoE) yielded the lowest affinity aptamer. However, other physicochemical factors such as size, structural complexity and intrinsic stability likely play important roles in governing protein-aptamer interactions. Further progress in the development of rapid selection techniques such as the microfluidic approach described here should make it possible to explore the relative impact of these target protein characteristics in greater detail. In parallel, recent work exploring the incorporation of non-natural bases into aptamers 
suggests additional means by which one might use selection strategies such as this to obtain molecules with yet higher affinity for an even broader range of protein targets.