A Noval Strategy For More Efficient Deoxyribozymes Based On 10-23 DNAzyme And Catalytic Mechanistic Studies-Chemical Modifications On The Catalytic Loop Of 10-23 DNAzyme With Protein-like Functional Groups

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 accompanyed by loss of activity. In our strategy with chemical modifications for more efficient deoxyribozymes, we started with the five-membered ring part of purines (A5, A9, A11, A12, A15, G1, G2, G6, G14) and 5-position of pyrimidines (T4, T8) in the catalytic loop of 10-23 deoxyribozyme. The results demonstrated that the all the 7-N atoms were necessary and 8-N atoms at A9 and G14 and G6 were more appropriately located for the catalytic activity. Further introduction of amino group and hydroxyl groups were much more beneficial for the activity. The modified 10-23 DNAzyme LKDZ21 and LKDZ14 were 11 and 6.3 fold faster than LKDZ01, respectively. From the effect of pH, divalent metal ions and cleavage site, we learnt that the modified DNAzymes have the same mechanism with 10-23 DNAzyme. These results proposed that the chemical modification at A9 and G14 with 8-aza-7-deaza-adenine and 8-aza-7-deaza-guanine analogues is a fascinating way to obtain more efficient modified 10-23 DNAzyme. The modification on T4 and T8 also demonstrated a potential site for more efficient deoxyribozymes. These studies demonstrated the catalytic potential of nucleic acids is enormous, which could be driven to that of protein enzymes by chemical modification and/or new in vitro selection technologies.