Applications Of DMSO-dependent DNAzyme In Environmental Analysis

RNA-cleaving DNAzymes（RCDs）are artificcial DNA catalyst that can catalytically cleave the RNA substrates in the presence of specific cofactors and generate amplificable signals,which enable them ideal biosensors for having both signal recognition elements and signal report units.Because of the ease of synthesis and modification,and having high stability,RCDs have great promising for biosensing in environmental monitoring,food safety and biological systems.Our group previously isolated an RCD from an random DNA library in 35%（V/V）dimethyl sulfoxide（DMSO）through in vitro selection.This DMSO-dependent RCD was further optimized to be a smaller one（t57r31）and the secondary structure thoroughly characterized as well.Based on these discoveries,the DNA catalyst molecule was engineered to respond specifically to heavy metal ions Hg²⁺and nucleic acid sequences of SARS-Co V-2 and influenza virus（Fluenza A）genomes in organic solvent environment.Laying the foundation for developing its application in environmental analysisMercury and its compounds are the most toxic heavy metals of concern in the environment。In order to construct a biosensor that specifically responds to Hg²⁺,a design scheme of transforming t57r31 cleavage into a"zippered"multicomponent DNAzyme（MNAzyme）was proposed in this thesis.It is hoped that MNAzyme can be activated by specific binding of Hg²⁺to thymine（T）to form T-Hg²⁺-T structure.Firstly,a"zipper"MNAzyme formed by double strands was designed and proven to be activated by the double strands.However,Hg²⁺was found not activate the MNAzyme with T-T mismatch sequences,but inhibite the cleavage activity of the RCD.The Hg²⁺-mediated inhibition showed a concentration-dose effect.We further investigated the RCD sequence,the cleavage substrate,metal ions,and p H values of the buffer.The data revealved that the p H value played an important role in Hg²⁺-mediated inhibition of the RCD.In order to understand whether the inhibition is specificial by Hg²⁺,different divalent metal ions(Zn²⁺,Co²⁺,Mg²⁺,Pb²⁺,Cu²⁺,Ca²⁺,Fe²⁺)were investigated.Besides Hg²⁺,Fe²⁺also strongly inhibited the activity of RCD,while Zn²⁺,Co²⁺,Pb²⁺,and Cu²⁺slightly inhibited the activity of RCD,but Ca²⁺had no effect on the activity of RCD.Although the MNAzyme for sensing Hg²⁺was not constructed,the toxic effect of heavy metal ions on RCD was identified,suggesting the catalyst poisoning caused by metal ions should be considered when using RCD for biosensing.The result also provides clues for studying the mechanism of DMSO-dependent RCD.The novel coronavirus（SARS-Co V-2）epidemic poses a great threat to human health and society.The cleavage sites of the DMSO-RCD were further profiled,and six positions were uncovered sutiable for the design of"zippered"MNAzymes.Among these spilitable positions,F site showed a longer target sequence requirement and faster catalytic rates.It is considered that a DNA sequence complementarily paired with the two parts of DNA strands at the splitted F position could activate the MNAzyme by a kind of"tape"adhesion,and thus to be used to biosensing the target sequence.In order to design the“taped”MNAzymes,we selected several 30-nt DNA sequences from the E,N and ORf1b genes of SARS-Co V-2 and influenza virus genome as targets.The MNAzymes were revealed to respond quickly to their targets without any“cross-talking”.The"taped"MNAzyme also responds to double-stranded DNA and RNA single strand targets.The target-activated"taped"MNAzymes are expected to apply in the specific detection of single-stranded RNA virus,micro RNA,as well as double-stranded DNAs,especially,for single-stranded RNA viruse,such as SARS-Co V-2,that is inactivated by ethanol or precipitated by ethanol might be directedly tested in this kind of organic solvents by employing the DMSO-dependent“taped”MNAzymes.Moreover,the strategy allows to avoid the tedious reverse transcription step in the traditional method for RNA virus detection,and might be applied as a point-of care test tool fot the detection of infectious RNA viruses in complex environment.In this thesis,the inhibition of the catalytic activity of DMSO-dependent RCD by heavy metal ions was investigated,and the data might provide clues for further study of the catalytic mechanism.Furthermore,the RCD was engineered for the design of“zippered”or“taped”MNAzyme.The“taped”MNAzyme further proven as general platform for biosensing nucleic acid targets in the reaction system containing organic solvents.This work expands the application of RCD sensors in environmental analysis and implies that the engineering region of the RCD is promising for introducing new recognition elements to construct smart responsive biosensors for the harmful water-insoluble molecules.