Application Of Purine Nucleoside Analogues In The Optimization Of 10-23 Deoxyribozyme Function

The 10-23 deoxyribozyme is a small catalytic DNA,it is capable of efficiently and specifically cleavage of target RNA.Numerous studies on RNA sequences,animals and clinical trials have demonstrated its great potential as genetic therapeutics.Different from antisense nucleic acid drugs of simple blocking or cleaving target RNA with RNase H,10-23 DNAzyme is a catalyst itself.Compared with siRNA drugs,its convenient and economic synthesis,as well as easy modification is a preferred practical choice.Therefore,higher catalytic efficiency is critical for the development of 10-23DNAzyme as one of genetic therapeutics.10-23 deoxyribozyme relies on Mg²⁺to exert catalytic activity,but the concentration of Mg²⁺in vivo is insufficient to exert high catalytic activity.Here,guanidinyl and imidazolyl groups were selected for the activation of DNAzyme through nucleoside analogues,because these two groups are often found to be active in protein enzymes.With new hydrogen bonding interactions of these two groups in the different positions within the catalytic core of 10-23 deoxyribozyme,more efficient DNAzymes will be selected.Four purine nucleoside analogs were synthesized,guanidinyl and imidazolyl groups were introduced at 6-amino group,8-position of adenine,and 7-position of 8-aza-7-deaza-adenine.By their corresponding phosphoramidites,24modified deoxyribozymes were synthesized with solid-phase synthesis protocol.Five adenines in the catalytic core（A5,A9,A11,A12,A15）and A0 of the recognition arm were substituted,respectively,with these four nucleoside analogues.The new interactions related to guanidinyl and imdazolyl groups in the context of the catatytic core are expected,due to their hydrogen-bonding ability and spacial occupation.Although their effect on the binding affinity of 10-23 DNAzyme toward target RNA is insignificant,the local conformational changes of the catalytic core induced by these exteral functional groups is very important for the catalytic ability of the modified DNAzymes,which is very interesting to be studied as the one of the approaches for more efficient DNAzymes.Previous researches showed that the 6-amino group of six adenines is an essential functional group that can affect the catalytic activity of 10-23 deoxyribozyme.In order to further optimize the function of 6-amino group,extra guanidinyl group with compound 12 and imidazolyl group with compound 14 were introduced in the six positions,respectively.At the A9 position,the deoxyribozyme HJCZ10 with a 3-fold increase in catalytic activity was obtained,and the activity was increased by 4 times for HJCZ22.This indicates that the 6-amino group of A9 is an appropriate position for modifications with increased catalytic activity of 10-23 deoxyribozyme.For the five-membered ring moiety of adenine,two modifications were conducted.At the 7-position of 8-aza-7-deaza-adenine,a guanidinyl group was introduced by compound 13,its substitution at A9 in DNAzyme HJCZ16 increased by 3 times of k_bos.The other modification site is 8-position,with compound 15,an imidazolyl group was introduced,but no deoxyribozyme with improved catalytic activity was obtained,indicating that the new interactions of imidazolyl group at this position is not favorable for the active catalytic conformation.All the substitutions of compounds 12,13,14,15 at other five positions in the catalytic core and A0 position were shown to be ineffective,it demonstrated that they are highly conservative residue for 10-23 DNAzyme.A9 is unique residue to be modified for more efficient DNAzymes.