Repair Enzyme-operated DNA Nanotechnology For Gene Detection And Imaging

Cancer has become one of the major disease threatening human health.The tumorigenesis and development of tumor are closely related to gene mutations and abnormal expression level.Gene mutations at specific sites and abnormal gene expression levels have become effective biomarkers,which play an important role in early diagnosis.The existing detection methods suffer from low specificity,poor sensitivity,and complicated design.Therefore,the development of highly selective genetic detection methods has become an urgent research content for the early molecular diagnosis.In this dissertation,inspired by the intracellular BER pathway,combined with the advantages of DNA nanotechnology in biosensors,and utilizing the new properties of nucleic acid repair enzymes,four advance and accurate gene imaging and single nucleotide mutation detection methods were developed.First,for the first time we successfully constructed a "burnt-bridge" DNA motor powered by APE 1,an endogenous enzyme responsible for DNA damage repair.Thanks to the high specificity of APE 1 to the AP site in double-stranded DNA,directional,autonomous movement was readily achieved with high controllability and processivity.The mobility of the DNA motor was studied by fluorescence approach and single-molecule microscopy,revealing APEldependent speed.The DNA motor can be operated along one-dimensional track in living cells without any external driving force,which mimics the BER pathway.Since APE 1 is believed to be a promising therapeutic target for cancers,this DNA motor,as a proof of concept,holds great promise for developing cellular diagnostic tools,gene regulators for DNA repair,and enzyme-mediated drug delivery.Then,a DNA circuit equipped with intracellular enzyme provides a unique approach for fluorescence imaging of miRNA in live cells allowing for in situ signal amplification.The DNA circuit for intracellular miRNA imaging consists of a hairpin strand,a reporter strand,and an amplifier APEl.It has the advantages of no signal leakage,1:n signal amplification,adjustable amplification strength,and the detection sensitivity is as low as 0.2 nM.The biomarker miR-21 in three cancer cells was in situ detected with remarkable sensitivity.Furthermore,the amplification strength can be precisely tuned by introducing the chemicals that affect the expression level of APEl.Our generalizable design holds great potential to be extended to non-nucleic acid targets and can potentially find applications for DNA computing,dynamic nanotechnology,and molecular diagnostics.Next,a novel method has been developed for the selective detection of SNM.By taking advantage of the combination of TSD and Endo Ⅳ catalyzed hydrolysis,we are able to detect all types of SNMs with high selectivity.This method has high selectivity for all 24 types of mutation including insertion and deletion.Among them,A>C,C>X,G>X and insertion achieve absolute discrimination,and the average discrimination factor for the other mutation is 491 times.SNM at low abundance down to 0.5%can be detected.The reaction mechanism was verified by single molecule fluorescence analysis.Both single molecule analysis and fluorescence measurement in bulk solution suggest that the branch migration in TSD can be accelerated by Endo Ⅳ through conformational selection pathway.This holds great potential for tuning the DNA assembly rate and would find broad applications in the fields of DNA structure and dynamic nanotechnology.In combination with PCR,BRAF V600E(c.1799T>A)mutation can be detected from A375 cancer cell lines whereas the corresponding wild-type from normal cell lines shows negligible fluorescence response.As a consequence,owing to the merits mentioned above,this approach would find broad applications in designing DNA nanotechnology based biosensors and POC diagnosis.At last,the single-nucleotide selectivity of hybridization based probe was greatly improved by utilizing the transient binding of short probe and disserting the binding kinetics.We constructed Gamma distribution model for state dwell time and optimization of assay conditions.The major advantage of our single molecule platform is its unique ability to resolve kinetic rates of binding by individual units in a digital manner.This allows us to directly identify the SNV at single molecule level and achieve SNV detection down to 0.01%mutant fraction.Utilizing this methord,KRAS c.34 G>A mutation in A549 cell line and BRAF c.1799 T>A mutation in A375 cell line can be detected.Looking ahead,the transient binding tag can become a promising signal reporter in single molecule analysis for heralding broad applications.This dissertation widens the application of nucleic acid repair enzymeassisted DNA nanotechnology.A series of high-efficiency and accurate gene detection methods were developed,which have the advantages of novel design,rapid reaction rate,high accuracy and sensitivity.These advance approaches may help to clinical diagnosis and precision treatment.