Chemical Modification On The Purine Residues In The Catalytic Loop Of 10-23 Deoxyribozyme And Catalytic Activity Evaluation

10-23 DNAzyme is a RNA-cleaving single-stranded DNA molecule selected by SELEX. Under simulated physiological conditions, it can cleave any purine- pyrimidine (RY) junction with high efficiency and sequence-specificity. Its application in vivo is limited because of their liability to nucleases digestion and lower physiological concentration of Mg2+. Chemical modification was used as an important solution to the challenge. Increased stability has been realized, but accompanied by loss of activity. In our strategy with chemical modifications for more efficient deoxyribozymes, we started with the five-membered ring part of nine purine residues (A5, A9, A11, A12, A15, G1, G2, G6, G14) in the catalytic loop of 10-23 deoxyribozyme. Their nine 7-nitrogen atoms are supposed to take part in the complex interactions responsible for the building of the catalytic conformation of 10-23 DNAzyme. Our research on the four dG residues with 7-deaza-2'-deoxyguanosine demonstrated that the all the four 7-N atoms were necessary for the activity. A more positive role for this nitrogen atom was explored with 8-aza-7-deaza-2'-deoxyguanosine, from which 8-nitrogen atoms at G2 and G14 were more appropriately located for the catalytic activity. Further introduction of hydroxyl group by 7-hydroxylpropy-8-aza-7-deaza-2'-deoxyguanosine at G2 and G14 resulted in a faster cleavage of LKWQ11 and LKWQ13.Based on our previous research on five dA residues, A9 was selected as the position for the positive roles of 7-substituted 8-aza-7-deaza-2'-deoxyadenosine analogs, especially, LKDZ21 with 7-aminopropyl-8-aza-7-deaza-2'-deoxyadenosine at A9 is 12-fold faster than the parent 10-23 DNAyzme. A structure-activity relationship was conducted on this structure. (1) The less positive effect of 7-aminopropyl-7-deaza-2'-deoxyadenosine at A9 of 10-23 DNAzyme indicated that the amino group and the 8-nitrogen atom in 7-aminopropyl-8-aza-7-deaza-2'-deoxyadenosine were cooperative in rate enhancement. (2) The incorporation of 7-aminopropynyl, 7-hydroxypropynyl, phenyacetyl substituted 8-aza-7-deaza-2'-deoxyadenosine analogs at A9 demonstrated the important influence of the linkage, the flexible linkage permits more freedom for the functional groups to induce a more favorable conformational change. (3) A larger functional group at A9 is more beneficial for the favorable conformational change, although it is not so powerful than other interactions like hydrogen bonding. (4) The incorporation of 7-aminopropynyl, 7-hydroxypropynyl, phenyacetyl substituted 8-aza-7-deaza-2'-deoxyadenosine analogs and 7-aminopropyl-7-deaza-2'-deoxyadenosine at other dA positions further demonstrated the unique and most potent A9 position for the lead structure 8-aza-7-deaza-2'-deoxyadenosine. (5) The effect of 6-amino group of five dA residues were screended with purine-2'-deoxyadenosine, the results informed us new positions for more potent 10-23 DNAyzme analogs.Thermal stability and CD spectra of the modified 10-23 DNAzyme analogs indicated that such kind of chemical modifications did not change the binding affinity of DNAzyme to the substrate and whole conformation. The observed rate enhancement is intrinsic for the DNAzymes, which is completely different from those by increasing binding affinity. However, these two methods could be combined for a more rate enhancement as well as enzymatic stability.Our results indicated that the catalytic potential of 10-23 DNAzyme could be further explored by chemical modifications. Practical applications of 10-23 DNAzyme will be undoubtedly developed by further optimization with this strategy. Functionalization on nucleic acids is a powerful tool for new functions of nucleic acids.