Fig. 2. Structural and activity analysis of single-active-site TSDzymes. (a) CD spectra of free hemin-G, TSDzyme (out) and TSDzyme (in), all at 2.0 μM; the peak at ∼263 nm is characteristic of the formation of a parallel G-quadruplex, and indicates that the scaffold-bound hemin-G retains the parallel G-quadruplex conformation adopted by the isolated catalytic domain. (b) The pK_a values of free hemin-G, TSDzyme (out) and TSDzyme (in) are identical, suggesting that the degree of ionisation of a catalytically important water molecule in the catalytic domain is unchanged between the three. (c) Catalytic activity depends on the placement of the hemin-G active site. It is shown that K_cat of the free hemin-G (10.6 ± 0.9 min^–1) increases 3-fold (to 30.2 ± 1.3 min^–1) when the hemin-G is incorporated outside the tetrahedral scaffold; meanwhile, it increases 4-fold (to 42.7 ± 1.5 min^–1) when it is incorporated inside the scaffold. (d) The tetrahedral scaffold also enhances the catalytic activity of other DNAzymes, such as EAD and B7.