New Functional Molecules Built On Nucleic Acid Structures With Catalytic Activity Of Nucleases

The reproduction of structure-activity relationship of biomolecules on a relatively simple and controllable molecular skeleton, such as enzyme mimicking, is a challenging task. But a successful strategy is not only helpful for the research on the mechanism of macromolecules, as well as their interactions with other molecules ( e.g., the interactions between nucleic acids and protein, enzymes and their ligands, antigen and antibody, receptors and acceptors), but also for the development of the new functional molecules as therapeutic agents and research tools in gene research and biotransfer.Various unique functions of biomolecules are derived from their specific structures. For enzyme families, different reactions are dependent on their different catalytic centers. While similar catalytic reactions and catalytic centers are found within a family of enzymes. As a result, in the research on artificial enzymes, the structural mimicking of catalytic centers of natural enzymes was fully explored, although the catalytic efficiencies of artificial enzymes was seldom up to the level of natural enzymes, anyhow, structure-activity relationship was completely demonstrated.DNA could be an ideal building block in the mimicking of bioactive structures due to its highly regular and rigid and reproducable structural features. Even with modified residues, the special positions of all the functional groups could be roughly expected. In DNA duplex or triplex, by incorporating functional group-bearing residues, a functional group assembly could be organized according to the active center of a biomolecule, if there is little influence of the modified residues on the duplex or triplex stability and conformation. With optimization of a special positions, including the positions of modified residues and the linkers for functional groups, a real copy of bioactive center would be mimicked on DNA helix, and new functions would be possibly assigned to the new structure.DNA helix is especially appropriate for mimicking nucleases. On the one hand, the catalytic DNA sequences could be complementary for the target DNA or RNA sequences, and then highly specific catalytic hydrolysis on the target DNA or RNA will be realized, on the other hand, the helix formation between the complementary catalytic sequence and the target sequence will bring the catalytic center and the phosphodiester closely enough to exert cleavage reactions.In our research DNase I and RNase A were mimicked on DNA triplex or duplex. With the help of the computer-aided molecular dynamic calculation, rational assemblies of functional groups on DNA structures were constructed. The catalytic functional groups, hydroxyl, imidazolyl, amino, and carboxyl groups were firstly attached to the nucleosides, and a series of modified phosphoramidites were synthesized for modified DNA synthesis. These functional groups will make up the active center of DNase I or RNase A with the formation of modified DNA duplex or triplex.The modified DNA sequences with catalytic functional groups were termed as functional sequences. The functional sequences could be complementary with target RNA sequences, acting as a new kind of catalytic antisense hydrolytic agent; they also could be be one or two of the three sequences in a triplex, as a new type of antigene tool. In a word, these structurally functionalized and bioactive nucleic acids might consist of a new kind of artificial biomolecules.Within duplexes, it was observed that all the functional sequences we designed (LK-XL-OP-009,LK-XL-OP-013,LK-XL-OP-009H,LK-XL-0P-013S,LK-XL-0P-013H) could cleave the complementary RNA phosphodiester bond efficiently, although their hydrolytic effects were different. LK-XL-OP-009 and LK-XL-OP-013 was functionalized with one amino group; LK-XL-0P-013S with one amino and one hydroxyl group, and LK-XL-0P-009H and LK-XL-OP-013H with one amino and one imidazolyl group. All the amino-containing functional sequences are active as cleavers of RNA phosphodiester bond, which is in contrast to the observation that mono-amino system is inactive in the hydrolysis of RNA.According to our observation of the pH- and Mg²⁺-dependence of catalytic hydrolysis of the functional sequences, it was possible that the amino group acted as Lewis base to promote the nucleophilic attack of 2'-oxygen atom on phosphodiester bond, and Mg²⁺as the Lewis acid, facilitating the leaving of 5'-oxygen atom. The hydrolytic efficiency of the catalytic system amino-Mg²⁺in Tris-HCl buffer was much higher than diamine and amino-imidazolyl system.Furthermore, it was demonstrated that LK-XL-OP-009, LK-XL-OP-013 and LK-XL-OP-013S hydrolyzed the target sequence with high site-selectivity, although more evidence would be needed to testify this observation. More importantly, LK-XL-OP-009, LK-XL-OP-013, LK-XL-OP-013S were catalytic functional sequences, multiple turnover was realized with them, according to our prelimilary experiments. It seems that the catalytic center of RNase A was successfully mimicked in DNA duplex. Interestingly, it is the necessary duplex formation with complementary target sequences that a catalytic center could be formed and hydrolysis of RNA phosphodiester bond conducted. This kind of catalytic property of complementary functional sequence could be potentially applied in the development of catalytic antisense agents and probes for RNA structure and its interactions with other molecules.Within modified triplex, one and/or two functional sequences could not make up an efficient catalytic center similar to DNase I, more assemblies of functional groups and reaction conditions are being optimized.