Another implication of this model would be that pre-bent DNA could be a better substrate for A3A binding, as A3A would not have to pay the entropic cost of bending the DNA. This bending of DNA could be achieved either by the inter-DNA interactions modeled in Fig. 5f, or when within a loop of a hairpin. To determine the significance of the bent DNA structure in the mechanism of A3 binding, we tested A3A affinity to a target deoxycytidine in the loop region of a DNA hairpin. The hairpin sequence was based on a previously identified potential RNA substrate for A3A, from succinate dehydrogenase complex iron sulfur subunit B (SDHB)35. The affinity for TTC in the loop region of this hairpin DNA was higher than that in linear DNA (26 nM vs 90–127 nM respectively). As expected, A3A had a higher affinity for the DNA hairpin with loop region containing TTC compared to one with AAA (26 nM vs ~676 nM respectively) (Fig. 6a). Interestingly, the Kd value for the hairpin (26 nM) is comparable to that for a single C in a polyT background (35 nM)28. This may imply that the Poly T DNA adopts a hairpin structure in solution, as has been reported36.Figure 6A3A specificity for substrate in loop region of stem-loop (hairpin) nucleic acids. Fluorescence anisotropy of TAMRA-labeled hairpin DNA and RNA binding to A3A(E72A). (a) Binding of A3A to a DNA version of the hairpin SDHB RNA containing TTC (dark blue) and AAA (light blue) in the loop region. (b) Binding of A3A to hairpin SDHB RNA (dark orange) and the same RNA sequence replacing the UC with AA in the loop region of the hairpin (light orange).