FIGURE 1.

(A) Secondary structures of the parent ligase, the pool of sequences used for selection, and the improved ligase (clone 23). Secondary structural elements (common to all three) are labeled on the pool. Joining regions (J) are named for the two paired regions (P) they connect. Residue numbering is with respect to the ligation junction, with the 5′-terminal residue of ribozyme assigned +1, and the 3′-terminal residue of the substrate assigned –1. All numbering is with reference to the improved ligase; insertions are marked with asterisks. Positions that were randomized in the pool are labeled N on the pool secondary structure; positions mutagenized at the 10% level are shown in lowercase. Sequences in gray on the pool secondary structure are, at left, the DNA portion of the substrate used for selection and, at top, the RT primer-binding site. (B) Improvement of pool activity over the course of the seven rounds of selection. Rates were measured at pH 6.0 in 10 mM Mg²⁺. (C) Crystal structure of the product of the improved ligase (Shechner et al. 2009). Elements of secondary structure are colored as in A.