Fig. 6. Amplification profiles of individual ribozymes in the continuous evolution mixture. Filled and open circles correspond to type I and type II ribozymes, respectively, that were isolated after transfer 15 of continuous evolution. The concentration of RNA was determined at various times, and the data were fit to the logistic equation: [RNA] = a/(1 + be^-ct), where a is the maximum extent of amplification and c is the exponential growth rate. Curvilinear regression coefficients were in the range of 0.998-0.990.

Fig. 7. Catalytic activity of the T80-1 ligase under continuous evolution conditions. Data were obtained from three independent experiments and fit to the Michaelis-Menten equation. Curvilinear regression coefficient was 0.954.

Fig. 8. Determination of the regiospecificity of ligation. Reaction of the T80-1 ligase with a 35-nt 5'-³²P-labeled substrate containing a single 3'-terminal ribonucleotide, followed by digestion of the ligated products with RNase T2, which cleaves 3',5', but not 2',5' linkages. Products of either alkaline or enzymatic digestion migrate slightly faster in a denaturing polyacrylamide gel compared to the unreacted substrate.

Fig. 9. Secondary structure analysis of the T80-1 ligase. N and D indicate native and denaturing conditions, respectively. Boxed areas indicate residues within the newly evolved accessory domain. (A) Enzymatic digestion of ligated ribozymes. Filled circles indicate residues accessible to cleavage in the native state: C89, C93, C98, U105, U110, and U114 (see Fig. 2B for numbering of residues). (B) Chemical modification of ligated ribozymes with either dimethyl sulfate (DMS) or kethoxal (KE), followed by reverse transcription. Open circle indicates residues protected from modification in the native state: G87, A90, G95, A96, G97, A99, G100, A101, G103, A104, A108, and G109. Note that chemical modification causes reverse transcriptase to terminate one residue before the corresponding position in the dideoxy sequencing ladder (lanes U, G, C, and A).

Fig. 10. Ligation activity of the T100-1 ribozyme and a mutant ribozyme that contains an altered sequence within the region of the 7-nt insertion. Both were tested with the truncated substrate (S⁰), a compensatory substrate that is complementary to the mutant ribozyme (S^M), an all-DNA version of the compensatory substrate (S^DNA), and an equimolar mixture of S⁰ and S^DNA. Time points were taken at 30 sec, 1 min, 2 min, 5 min, and 10 min. Reaction conditions mimicked those of continuous evolution except that the protein enzymes were omitted, the total concentration of substrate was 10 mM, and the ribozyme concentration was 1 mM.