Retraining and Optimizing DNA-Hydrolyzing Deoxyribozymes for Robust Single- and Multiple-Turnover Activities

Recently, we reported two classes of Zn2+-dependent DNA hydrolyzing deoxyribozymes. The class I deoxyribozymes can adopt a secondary structure of either hairpin or stem-loop-stem. The corresponding most active representatives, I-RI and I-R3, exhibit single-turnover k(obs) values of similar to 0.059 and similar to 1.0 min(-1) at 37 degrees C, respectively. Further analysis revealed that I-R3 could perform slow multiple-turnover catalysis with a k(cat), of similar to 0.017 min(-1) at 37 degrees C. In this study, we sought to retrain and optimize the class I deoxyribozymes for robust single- and multiple-turnover cleavage activities. Refined consensus sequences were derived based on the data of in vitro reselection from the degenerate DNA pools. By examining individual candidates, we obtained the I-RI mutants I-R1a-c with improved single turnover kobs values of 0.68-0.76 min(-1) at 37 degrees C, over 10 times faster than I-R1. Meanwhile, we further demonstrated that I-R1a-c and I-R3 are thermophilic. As temperature went higher beyond 45 degrees C, I-R3 cleaved faster with the k(obs) value reaching its maximum of min(-1) at 54 degrees C. Using a series of the k(obs) values of I-R3 from 37 to 54 degrees C, we calculated the apparent activation energy E-a to be similar to 15 +/- 3 kcal/mol for the DNA-catalyzed hydrolysis of DNA phosphodiester bond. In addition, we were able to design a simple yet efficient thermal-cycling protocol to boost the effective k(cat) of I-R3 from 0.017 to 0.50 min(-1), which corresponds to an similar to 30-fold improvement of the multiple-turnover activity. The data and findings provide insights on the enzymatic robustness of DNA-catalyzed DNA hydrolysis and offer general strategies to study various DNA enzymes.