Study On Chemical Modification And Activity Of Functional Nucleic Acid

Since the discovery of 10-23 DNAzyme,the researchers have conducted extensive research on it.Cell culture experiments have shown that 10-23 DNAzyme,as a potential drug,are effective at downregulating various genes of therapeutic significance,such as viral,cancer,cardiovascular or other related diseases;In vivo studies have shown that Dzs are highly target-selective,non-toxic and non-immunogenic gene-silencing agents.However,due to the low intracellular Mg²⁺and enzyme hydrolysis,the application is limited,and the catalytic conformation is not clearly defined.Thestrategy of chemical modification of 10-23 DNAzyme was modified to improve its stability and catalytic rate,thereby reducing the dependence of two divalent metal ion.At the same time,through the chemical modification,we can define the function of each functional group in the catalytic activity,and thus to study the catalytic conformation.On the basis of previous studies,we put7-?5-aminopentyl?-8-aza-7-deaza-2'-deoxyadenosine into A5 and A9 of 10-23deoxyribozyme catalysis center,and the activity evaluation show that amino link extension does not accelerate the reaction rate,contrarily,due to the steric hindrance of alkyl arm may lower catalytic rate,so the 3 carbon atoms long alkyl arm of the appropriate amine media should play a role.The 6-amino of adenine has hydrogen bonding capability,we speculate that in the catalytic domain of 10-23 DNAzyme,it is related to the conservation of the five adenine bases.In order to further explore on the role of 6-amino,we designed the compound 6-H-2'-deoxyadenosine based on adenine.Then,in order to obtain new modification sites and more efficient deoxyribozymes,we designed and synthesized three compounds N⁶-[?2-aminoethyl?]-2'-deoxyadenosine?N⁶-[?3-aminopropyl?]-2'-deoxyade- -nosine and N⁶-[2-?imidazole-4-yl?ethyl]-2'-deoxyadenosine,which retain the hydrogen bonds ability of 6-amino group,and external amino and imidazolyl groups were introduced.All these compounds were synthesized and converted to their phosphormidites for the synthesis of the modified DNAzymes.Enzymatic activity evaluation of the modified DNAzymes showed that the 6-amino of desoxyadenossine is very important in the DNAzyme catalytic domain,especially in the sites A5,A9,A11 and A12.Among them,A5 not only needs 6-amino groups of the hydrogen bonding interaction but also need a hydrophobic group;6-amino of A11 and A12 is very conservative and can not be changed;the role of 6-amino of A9 and A15 can be improved by chemical modification.In the isonucleosides-modified DNAzymes,we found that the catalytic conformation of 10-23 DNAzyme could be optimized.The results also showed that isonucleoside on the arms had different effect on the activity,the more close to the catalytic center,the more decreased of catalytic rate,especially when in the A0position,DNAzyme activity almost disappeared;When it was lacated at a position two nucleotides from the catalytic center,the catalytic activity of the prototype?DZ01?are basically the same.In further study,we investigated the conservation of A0 with compounds 1,4-8,16-18,the result showed that A0 is a conserved site,these modifications always led to a decrease of the rate of DNAzyme catalysis.But the selectivity of cleavage site of10-23 DNAenzyme and the divalent metal ion dependence did not change.The stability of U-dA0 base pairs is not only related to the DNA recognition of target substrate but also influence the complexes of enzyme-target,through the base stacking and the interactions with other residues in the catalytic core.