Aptamer HCR system. (a) Aptamer trigger mechanism. Binding of the DNA aptamer (blue) to ATP (37) exposes a sticky end (8) (magenta) that triggers the HCR mechanism of Fig. 1 by opening hairpin H2. The region x (red) is introduced to help stabilize the trigger in the absence of analyte (8). The region b^* includes both the hairpin loop and the portion of the stem complementary to x. (b and c) ATP detection via HCR. Agarose (b) and acrylamide (c) gels demonstrate amplification of ATP recognition, with the former providing better resolution of HCR products and the latter providing better resolution of unreacted species. Reactions are performed with 1.4 mM ATP and GTP and all DNA species at 1 μM. Lane 1: the hairpins do not polymerize when mixed (H1 + H2). Lane 2: addition of simple initiator triggers HCR (H1 + H2 + I). Lane 3: hairpins with ATP (H1 + H2 + ATP). Lane 4: aptamer initiator with ATP (I^ATP + ATP). Lanes 5 and 7: weak spurious HCR in the absence of ATP (H1 + H2 + I^ATP) or the presence of GTP (H1 + H2 + I^ATP + GTP), respectively. Lane 6: strong HCR amplification of ATP recognition (H1 + H2 + I^ATP + ATP). Lane 8: DNA ladder (100–1,000 bp in 100-bp increments).

Basic HCR system. (a–c) Secondary structure schematic of HCR function. Letters marked with * are complementary to the corresponding unmarked letter. (a) Hairpins H1 and H2 are stable in the absence of initiator I. (b) I nucleates at the sticky end of H1 and undergoes an unbiased strand displacement interaction to open the hairpin. (c) The newly exposed sticky end of H1 nucleates at the sticky end of H2 and opens the hairpin to expose a sticky end on H2 that is identical in sequence to I. Hence, each copy of I can propagate a chain reaction of hybridization events between alternating H1 and H2 hairpins to form a nicked double-helix, amplifying the signal of initiator binding. (d) Effect of initiator concentration on HCR amplification. Lanes 2–7: six different concentrations of initiator (0.00,10.00, 3.20, 1.00, 0.32, and 0.10 μM) in a 1 μM mixture of H1 and H2. Lanes 1 and 8: DNA markers with 100-bp and 500-bp increments, respectively. (e) HCR kinetics. The hairpin monomers do not hybridize before triggering by initiator [(H1^2AP + 1.2× H2) + 0.5× I, red]. The same quenched baseline is achieved without HCR by adding excess initiator to H1^2AP in the absence of H2 (H1^2AP + 4.0× I, green). Addition of insufficient initiator to H1^2AP provides only partial quenching (H1^2AP + 0.5× I, blue).