Enzymatic Properties And Structure Activity Relationships Of I-R3 DNAzyme

In the early 1980 s, Cech and Altman found RNA molecules with catalytic function in the intron of Tetrahymena and bacteria RNase P respectively, named RNAzyme(ribozyme). RNAzyme was found to break the traditional academic idea "enzyme is protein", which became an important breakthrough in the research of enzyme and directly promoted the research and development of DNA catalytic function. In 1994, Breaker and Joyce obtained single-stranded DNA molecules by in vitro evolution, named DNAzyme(deoxyribozyme), that have capability of catalyzing RNA cleavage. As a new member of the family of enzymes, discovery of DNAzymes was an important progress in the study of enzymes. So far, vast majority of DNAzymes have been obtained through in vitro evolution. DNAzyme catalysis can be divided into 3 types: cleavage, linkage and modification, wherein DNAzymes with cleavage activity are the most. Recently, DNAzyme exhibited higher application value and broad application prospect on gene silencing, DNA molecular device and biological sensor.In 2013, Breaker’s Lab obtained a series of Zn2+-dependent DNAzymes with DNA-cleaving activity by in vitro evolution, wherein I-R3 has the highest catalytic activity. The structure of I-R3 shows a “stem-loop-stem” form and the catalytic core is located in the "loop". The catalytic core is composed of 15 deoxynucleotides, the enzyme and the substrate recognition binding region is the "stem" structure. In this paper, we use I-R3 DNAzyme as the research object, mainly focused on four aspects, including enzymatic properties, structure-activity relationship, mutant A5 G and application.Based on the structure of cis I-R3 DNAzyme, we designed two kinds of structures: I-R3 N and I-R3 S. Our impact on the I-R3 N and I-R3 S DNAzyme catalytic activity were characterized in detail, including the conditions of Zn2+ concentration, thermal denaturation, NaCl concentration, KCl concentration, metal ion selectivity, anti metal ion interference, pH value and reaction time et al. The experimental results show that the catalytic activity of I-R3 N and I-R3 S is closely related to the concentration of Zn2+. The optimum Zn2+ concentration is 10 mM with its strict specificity. It shows the Mg2+ and Ca2+ anti-interference ability and thermal denaturation cannot enhance catalytic activity. NaCl and KCl inhibited the catalytic activity. And the pH in the reaction system has great influence on the activity, the optimum pH was 7.0, after 60 min of reaction, the catalytic efficiency tends to be stable.Under the optimum reaction conditions, the catalytic activity of I-R3 N DNAzyme is higher than I-R3 S, so we choose I-R3 N DNAzyme to structure-activity relationship study. Under different conditions(pH 6.0, 7.0 or 8.0), we compared the catalytic activity of the 57 mutants of the I-R3 DNAzyme. Then through the second round of mutation, clear relationship between catalytic activity and deoxyribonucleotide in catalytic core and determine the mutation hotspot and find high activity mutant A5 G. At the same time, the I-R3 N, I-R3 S and mutant A5 C cleavage sites were identified and analyzed in detail. Then we carried out the characterization of high activity mutant A5 G on the conditions of concentration of Zn2+, metal ion selectivity, anti-interference, pH value and reaction time.Finally, based on the principle of fluorescence resonance energy transfer, we chemically modified fluorescence and quenched molecules in the I-R3 N of 5’ and 3’ ends respectively, and by detecting the fluorescence intensity to directly determine the changes in its catalytic activity. On the basis of this result, we expect to develop a high sensitive Zn2+ fluorescence sensor, by the study of DNAzyme enzymatic basic research extended to enzyme engineering applications.