Functional Optimization Of Cleavage-type 10-23 DNAzyme And Its Application In Biosensor

10-23 DNAzyme is a DNA molecule with the ability to catalyze the cleavage of complementary RNAs.The enzyme consists of a 15-mer conserved catalytic domain and a substrate recognition arm at each end.Through Watson-Crick base pairing,the recognition arms hybridize with the substrate to form a localized double helix structures.The catalytic reaction of 10-23 DNAzyme conducts the catalytic reaction assisted by Mg²⁺.Based on the non-conserved feature of T8,it has been proposed that the catalytic domain was flexible enough to be split at T8,either between T8 and C7 or between T8and A9,from T8 and A9 or C7 a short sequence is added,by which the catalytic domain could be reunited by forming a complementary duplex with an extra sequence.This catalytic DNAzyme is called split 10-23 DNAzyme or MNAzyme.The extra sequence is called a nucleic acid initiator which reunites and starts the catalytic reaction of the MNAzyme.The concentration of the nucleic acid initiator has been demonstrated to be positively proportional to the signaling related to the cleavage reaction.Therefore,the nucleic acid initiator could be recognized as the the analyte of the MNAzyme,and it can be selected from a virus,bacteria or disease-associated DNA or RNA.Therefore,this catalytic reaction system has the potential to be developed into a general-purpose biosensor.Based on our research group’s research on the catalytic domain of 10-23DNAzyme,the Here,the conserved N gene of 2019-n Co V is used as the nucleic acid initiator to establish the detection method with the 10-23 DNAzyme-based MNAzyme.From the conserved N gene of 2019-n Co V（Ref Gene:MN908947）,the sequence29142-29338,which was demonstrated to be an accessible target by PCR,was used as the target in the design of MNAzyme.Six target sequences（29NT1,29NT2,29NT3,GOT1,GOT2,and GNT）were selected as the nucleic acid initiator for the MNAzyme,the target-recognition arms of MNAzyme were complementary each of them,respectively,therefore,six MNAzymes（MNAzyme01 to MNAzyme06）were designed.With the MNAzyme01（PSUB+29N1+29N2+29NT1）,it was demonstrated that each component was essential for the catalytic reaction.A comparison was made for cleavage sites between the C7 and T8 and between T8 and A9,and the latter formed a more active MNAzyme07.The two key factors for the catalytic reaction of 10-23DNAzyme was used for MNAzyme evalution,the p H of the buffer（Tris-HCl）and the concentration of Mg²⁺.The same p H-dependence and Mg²⁺concentration-dependence between 10-23DNAzyme and MNAzyme01 was observed.among them,MNAzyme02 and MNAzyme03 conducted the fastest reaction,under the conditions of p H 8.5（Tris-HCl）and 50 m M Mg²⁺.In the case of MNAzyme,a new substrate PSUB3 was designed for MNAzyme01和MNAzyme03,MNAzyme07 and MNAzyme09 were constructed,respectively,and they had a faster reaction.The detection limit of MNAzyme09 was 25 p M.Based on the reaction rate of six splitting MNAzyme,the best nucleic acid initiator 29NT3 was selected,and the splitting MNAzyme involved in the formation were named MNAzyme09.Based on MNAzyme09,under the conditions of p H 8.5 and 50 m M Mg²⁺,the detection limit was 50 p M.Although the limit is far from the real detection practice,it offers a core element for further optimization of the method,either by modification on the catalytic core or introduction of more sensitive analytical methods.With MNAzyme09,the catalytic core was studied for the conservation of several residues.Among the 15residues,the highly conserved T4 and G14 were selected for mutation with adenine.These results demonstrated that MNAzyme had the same catalytic core requirement as 10-23 DNAzyme.It was thus suggested that both adopt the similar catalytic conformation.Together with the same influence of the p H and Mg²⁺,it was concluded that MNAzyme and 10-23 DNAzyme conducted the catalytic reaction with the same mechanism,with different active conformations.Based on the mechanistic studies,A9 of MNAzyme09 and MNAzyme14 will be modified with deoxyadenosine analogue 1[7-（3-aminopropyl）-8-aza-7-deaza-2’-deoxyadenosine],and more efficient MNAzyme were expected,and this research is going on.More importantly,the target was studied for its conservation in the MNAzyme system.With MNAzyme09,mutations were introduced at different positions far or near the cleavage site.The mutations near the cleavage site were generally detrimental for the reaction,while the mutations far from the cleavage site induced much less effect on the reaction.This fact demonstrated the flexibility of MNAzyme-based method.With a highly conserved target nucleic acid sequence from the disease-related RNA/DNAs or that of bacteria or viruses,the mutation could be designed in the positions far from the cleavage site for a general detection method for a family of nucleic acids,or the mutation could be located near the cleavage site,the specific mutation or strains of bacteria/virus could be detected.Based on MNAzyme09,miR21 was detected with MNAzyme14,in which miR21 was used as the target sequence.The detection limit is0.5 n M under present conditions,with a linear range of 20 n M-0.5 n M.In addition,three kinds of MNAzymes or DNAzymes were developed based on T-Hg-T basepair.The first was constructed on MNAzyme17,in which a T-rich motif was inserted in the target-recognition arm and the target sequence in three MNAzymes.The formation of T-Hg-T basepair is helpful for their reactions,but the detection limits need to be increased for better performance.It is worthwhile to be noted that the random interaction between Hg²⁺and all nucleobases are not beneficial for the catalytic conformation,which led to a counteraction of the positive contribution of T-Hg-T,as demonstrated by DNAzyme DZ02.The second formula is designed by two T-rich motifs from the cleavage site T8 and A9,respectively,six structures were constructed,in terms of different length of T-rich motifs,and no target sequence was required for the catalytic core formation.Unfortunately,no reaction was observed under present conditions.The third design was based on 10-23DNAzyme,six structures were formed by the insertion of T-rich motifs between T8and A9.Onl7 ADZ27 showed the contribution of T-Hg-T basepair,it could be further optimized for Hg²⁺detection.In this paper,the research of efficient split 10-23 deoxyribozyme is carried out.The study of its struction-activity relationship provides the core components for its biosensor application,which has important scientific and practical significance.