Key Residues Grafting Yields Novel Deoxyribozymes

Over the past decade, a variety of deoxyribozymes (also referred to as DNAzyme or catalytic DNA) that can catalyze a broad range of chemical reactions have been developed by directed evolution. Some of them have been improved specific characteristics and functionsthrough rational design, such as regulation of catalytic activity,deoxyribozyme-based sensor, and construction of molecular logic gate.However, the design of a deoxyribozyme with new catalytic activity remains a challenge due to a lack of understanding ofstructure-function relations. Recently, the rational design of protein with novel functions and properties has generated significant progress,these strategies of which, including key residues grafting, can also be applied to the design of deoxyribozyme. In this report, we used key residues grafting to generate a new catalytic activity within a DNA scaffold.In the first part of study, the key deoxyribonucleotide residuesassociated with catalytic activity in the known two types of Mg2+-dependent deoxyribozymes (8-17 deoxyribozyme and 10-23 deoxyribozyme) that carry out the cleavage of an RNA had to be identified in the first instance. By comparing their catalytic domains, the K (5'-AGC-3') and L (5'-ACGA-3') sequences and the g·T wobble pair were selected to act as the grafting key deoxyribonucleotide residues.In selecting a DNA scaffold, the hammerhead ribozyme (HRZ) can act as an ideal starting point. The hammerhead ribozyme, originally founded in small RNA satellites of plant viruses, is a small RNA motif that performs a site-specific phosphodiester bond cleavage reaction. Here,we prepared a hammerhead ribozyme as a DNA molecule, referred to as the hammerhead DNA scaffold. Its secondary structure was analogous to that of the 8-17 deoxyribozyme, both of which displayed a stem-loop structure.We designed the four projected structures, HL1, HL2, HL3 and HL4, respectively, which the key residues were grafted within the unconserved region of the DNA scaffold. HL1 and HL2 were shown to catalyze the Mn2+-dependent cleavage of an RNA substrate, and to cleave the substrate at the g/uag site, not at the presumed gua/g cleavage site, namely hammerhead-like deoxyribozyme. However, the HL3 and HL4 showed no detectable catalytic activity. To fine-tune the activity of hammerhead-like deoxyribozyme, we set a series of mutations to HL1, in which M3 (T2.1→G2.1) had the highest activity, the kobs of which is 4.8×10-2 min-1, about 9.3 fold greater than the kobs(5.1×10-3 min-1) of HL1. Subsequently, catalytic behavior of the hammerhead-like deoxyribozyme HL1 and M3 were identified. Under single-turnover conditions, analysis of the pH dependence of the M3 catalyst revealed that it had appreciable activity only in a narrow pH range 7.6-9.0, with maximum activity observed at about pH 8.2. The optimal pH 8.2 of M3 is modestly higher than the optimum pH 7.8 of the HL1 deoxyribozyme. The metal ion Mn2+ is necessary for the hammerhead-like deoxyribozyme catalysis. Over a range 0.5-50mM of Mn2+ concentrations, the reaction rate for HL1 and M3 cleavage was maximal in the presence of 10 mM Mn2+. The above results indicated that pH and metal ion concentrations can obviously affect the catalytic activity.After characterization of the hammerhead-like deoxyribozyme, the M3 mutant with the highest catalytic activity was converted to the corresponding RNA (RM3). The activity experiments showed that RM3 had the RNA-cleaving activity of hammerhead ribozyme in the presence of a number of different divalent metal ions. The metal ion cofactors decreased in efficiency (Mn2+>Mg2+>Ca2+>Co2+), and Cu2+, Zn2+ and Ba2+ ions were unable to support the cleavage activity of RM3, which was the same trend observed for HRZ. . The optimal pH of RM3 is 7.8, similar to that of HRZ (pH 7.5). The kinetic characterization of RM3 and HRZ hammerhead ribozymes under single-turnover conditions revealed that the kobs (5.6×10-2 min-1) of RM3 is lower than that (7.8×10-1 min-1) of HRZ, demonstrating that the u→c change at ribonucleotide position 7 and replacement of the former loop sequence 5'-guga-3'by 5'-agc-3'considerably influences the cleavage activity of hammerhead ribozyme.In the second part of the study, we prepared pistol-like deoxyribozyme as a DNA scaffold and put key residues of 8-17 or 10-23 deoxyribozymes into the unconserved region of the DNA scaffold, such as stem I, II and loop I, II. Of the thirteen projected structures, the activity experiments showed that the DT3 had both appropriate DNA- and RNA-cleaving activity which was a deoxyribozyme with double-catalytic functions. At present, we have charactered properties of the deoxyribozyme DT3, and compared with the"parent"pistol-like and 8-17 deoxyribozymes.When catalyzing DNA substrate, DT3 deoxyribozyme has shown a strictly Cu2+-dependent cleavage. The DNA-cleaving activity was maximal in the presence of 100 uM Cu2+, which was 10 fold greater than that of pistol-like deoxyribozyme. The catalytic activity of DT3 and pistol-like deoxyribozymes slowly increased at a pH range from 3.0 to 10.0, and had no obvious influence at a temperature range from 12 to 48°C. The most different property between DT3 and pistol-like deoxyribozymes was the cleavage sites of DNA substrate. The DT3 had the two cleavage sites and pistol-like had the four.When catalyzing RNA substrate, the properties of DT3 deoxyribozyme were compared with those of 8-17 deoxyribozyme. The optimal Mg2+ concentration of DT3 and 8-17 deoxyribozymes was at 1 mM. The catalytic activity of both deoxyribozymes had no obvious change at a pH range from 3.0 to 10.0, and increased as a pH rose from 3.0 to 10.0, but the higher temperature can disrupt the RNA substrate.Taken together, we obtained two new types of deoxyribozymes through key residues grafting, referred to as hammerhead-like deoxyribozyme and double-enzymatic deoxyribozyme, respectively. The results provided a novel approach in constructing deoxyribozymes by rational design, with potentially general significance. The construction of hammerhead-like deoxyribozyme demonstrated that a new catalytic activity can be generated within a DNA scaffold by rational design. The special feature of the deoxyribozyme has achieved that deoxyribozyme and ribozyme can be converted into each other, which provided evidence for the transfer of catalytic function between different nucleic acid-like molecules. The obtained double-enzymaticdeoxyribozyme realized a goal of"one DNA scaffold, two enzymatic functions". The DNA of the deoxyribozyme was probably a"divergent sequence"generated from evolution of pistol-like or 8-17 deoxyribozyme function. The combination of the aboved results and the study of"ribozyme encoded ribopepzyme"can outline the transfer of catalytic function between biological macromolecules, which can provide a new clue for the origin and evolution of enzyme.