Endonuclease Ⅳ-assisted Strategies For Low-abundance DNA Point Mutation Detection

As an essential clinical biomarker,DNA mutation is closely related to the occurrence and development of various diseases,especially cancer.DNA mutations occur in different types,including nucleotide substitutions,nucleotide insertions,deletions,etc.Singlenucleotide substitution mutation(DNA point mutation)is the most common type among them.Since there is only one nucleotide difference between the mutant-type target(MT)and the wild-type target(WT),it is difficult to distinguish them due to ignorable physical or chemical signal changes.In addition,in the early stage of cancer,MT is usually surrounded by numerous WT,resulting in a low abundance,further increasing the detection difficulty.However,currently developed low-abundance DNA point mutation detection strategies require complex sequence design,tedious temperature optimization,or rely on expensive analytical instruments,which limits their clinical applications.In order to overcome the above limitations,Endonuclease Ⅳ(Endo Ⅳ)is selected as the difference amplifier between MT and WT.A series of simple,high-sensitivity and lowcost DNA point mutation detection methods without temperature optimization were developed and applied to detect clinical epidermal growth factor receptor(EGFR)-related mutations.Research methodsIn this dissertation,based on our understanding of the underlying mechanism of Endo Ⅳ hydrolysis of the apurinic/apyrimidinic(AP)site in the double-stranded or singlestranded DNA,appropriate AP probe strands modified with fluorescent and quenching groups and the related target strands were designed.Target strands were designed to hybridize with AP probe strands to form different double-stranded DNA structures.The fluorescence analysis method was used to find the effect of Endo Ⅳ on the target strand and AP site.The hydrolysis activity rules of Endo Ⅳ to the different structural substrates were applied to detect DNA point mutation.Research resultsThe main results of this dissertation are as follows:(1)The side activity of Endo Ⅳ hydrolysis of AP sites in the single-stranded DNA adversely affects the Endo Ⅳ’s use in the detection of DNA point mutation.Our study found that Endo Ⅳ’s side activity is mainly related to the transient intramolecular or intermolecular binding due to the flexibility of single-stranded DNA.The side activity of Endo Ⅳ can be suppressed by adding an extra strand that hybridizes with the single-stranded portion of AP.After the side activity of Endo Ⅳ was inhibited,the discrimination factor of Endo Ⅳ between the mutant and wild-type strand was increased by about 20 times.(Chapter 2)(2)Breaking the traditional cognition that the optimal substrate of Endo Ⅳ is a fully matched doublestranded substrate,it was found that Endo Ⅳ has the highest hydrolytic activity to the recessed double-stranded substrates with only one or three bases at the 3’ downstream or 5’upstream of AP site.In addition,these unique substrate structures enhanced Endo Ⅳ’s ability to discriminate single-base mismatches.Combined with lambda exonuclease,deoxyribozyme,and asymmetric polymerase chain reaction,a 0.01% mutant-type target could be distinguished.(Chapters 3 to 5)(3)Human apurinic/apyrimidinic endonuclease 1and two common restriction endonucleases(Nt.Bsm AI and Nt.Bst NBI)also have similar substrates preferences like Endo Ⅳ.(Chapter 6).ConclusionsA series of simple,general DNA point mutation detection strategies were developed and successfully applied to distinguish various clinical related DNA point mutations.In addition,the method for exploring the properties of Endo Ⅳ in this dissertation can also be applied to explore the new properties of other enzymes.Finally,the shortcomings and some directions for future work were also proposed.Overall,this dissertation provided specific ideas and assistance for developing new DNA point mutation detection strategies.