Fig. 3. We reengineered the parent aptamer to produce higher-gain E-AB signaling. (A) We did so by destabilizing the aptamer's stem-loop (thus increasing the population of unfolded molecules poised to respond to target) via either introduction of one (CA40_1MM) or two (CA40_2MM) mismatches or via truncation (CA36, CA32, CA28, CA16) of the stem. (B) When challenged in a simple buffered solution all of the re-engineered variants exhibited higher gain than that of the parent aptamer (see ESI Table 1†), with the most destabilized (CA40_2MM, CA32, CA28, CA16) producing the greatest signal gain. (C) When tested in whole blood their gain and affinity are reduced, but the best performing nevertheless still support high-gain E-AB sensing.
