A Novel Hammerhead Deoxyribozyme Is Obtained By Rational Design

Ribozyme (or RNAzyme) is a segment RNA that can act as a catalyst. Thediscoveries of Ribozyme enrich and develope the conception of Enzyme. In1994, Joyce et al. obtained a single-stranded DNA with the catalytic activitythrough in vitro selection, which is deoxyribozyme or DNAzyme.At present, all obtained deoxyribozymes are acquired through in vitro selection,and no novel deoxyribozyme obtained by rational design is reported. In thisreport, we would in here describe a novel deoxyribozyme obtained by rationaldesign. First, we compared with the structure and sequence of 10-23deoxyribozyme and 8-17 deoxyribozyme and find the highly conservativenucleic acid sequences (E, F) in the catalytic domains of 10-23 deoxyribozymeand 8-17 deoxyribozyme. Then we take the DNA of hammerhead ribozyme asthe scaffold (hammerhead scaffold), and graft the conservative sequences onthe catalytic domain and the loop respectively, finally we may obtain a noveldeoxyribozyme ――hammerhead deoxyribozyme.Based on different considerations, we adopt four kinds of schemes to designhammerhead deoxyribozymes which are HD-1, HD-2, HD-3 and HD-4,respectively (Fig. 3.3).On the design of the cleavage site of substrate,we use the cleavage site ofhammerhead ribozyme 5'-GUX-3' as the reference, on the other hand weconsider the cleavage site of 8-17 deoxyribozyme 5'-AG-3'. Based on both,we design 5'-GUAG-3' as the cleavage site of hammerhead deoxyribozymeand speculate that the site of cleavage is between A and G..The experiment results indicate that HD-1 and HD-2 have the ability to cleavethe substrate, while HD-3 and HD-4 have no catalytic activity (Fig. 4.1). Andenzymology characters of hammerhead deoxyribozyme HD-1 indicate thecleavage site of HD-1 is between G and U, not A and G (Fig.4.1). Theoptimum temperature is 37℃, the optimum pH is 7.8. and the optimum Mn2+concentration is 10mM, In addition, we find that in the condition of 30℃ andCo2+, HD-1 and HD-2 also have weaker catalytic activity(Fig.4.5).As far as the relationship between the structure and the function, first we doresearch on rG·dT which effects on the catalytic activity. Experiment dataindicate rG·dT is very important for the catalytic activity. If there is no rG·dTor relative structure, the catalytic activity decrease deeply or lost, And whenthe rG·dT is changed into rG dG, the catalytic activity increase obviously.Subsequently, we research on the Stem-loop which effects on the catalyticactivity of hammerhead deoxyribozyme. The results indicate the stem-loop haseffect on the catalytic activity, when the stem is less than four, or if the Esequence in the loop is replaced by other sequence, the catalytic activitydecreases deeply.In the condition of single turnover ( Enzyme/Substrate=40:1),we do kinetics ofenzyme-catalyzed reactions analysis on HD-1 and its mutants M1, M2, M3.The Kobs of HD-1 is 0.0051;Kobs of M1 is 0.00495 which is close to the Kobs ofHD-1;the Kobs of M2 is 0.00183 which is less than the Kobs of HD-1;whilethe Kobs of M3 is 0.04765 which is about as much as 9.3 fold of HD-1.Through the above researches, we think, on the design of deoxyribozyme wecan use the catalytic domains and characters of other ribozymes anddeoxyribozymes for reference, rationally design various noveldeoxyribozymes. When the active site is imported, we can consider loopimplant and the experiments have verified this strategy is feasible andeffective.The structure and sequence of hammerhead deoxyribozyme and hammerheadribozyme is similar, only have the difference in the non-conservative domains.If the experiment results indicate the RNA of hammerhead deoxyribozyme hasthe activity of hammerhead riboyzme, we will realize the conversion ofribozyme and deoxyribozyme.This research is part of the research of "Enzyme Conversion", and integratewith other parts of "Enzyme Conversion", we propose an early life moleculesevolution hypothesis. Compare to the "RNA world", this hypothesis candescribe the relationship of life molecules in the early evolution more roundlyand more definitely. At the same time, it deeps and continues our lab previousresearches on the ribozyme and deoxyribozyme.