Design Of Novel Functionalized Nucleic Acid Probes And Its Biosensing Application

Biosensing and bioanalysis is to convert target recognition into measurable signals through specific recognition elements and signal transduction methods,so as to achieve the assessment of biomolecules content or function.It have not only advanced the scientific research of analytical chemistry and life science,but also promoted the development of clinical diagnosis that bases on changes in molecular characteristics.Nucleic acid probes have been widely constructed and used in biosensing and bioanalysis,due to their diverse recognition formats,flexible signal transduction and signal output methods.Among the signal output methods,fluorescent methods have attracted much attention because of their high sensitivity and convenience for in-situ analysis of intracellular biomolecules.Here,we focus on the design of functionalized nucleic acid probes and the construction of novel fluorescence biosensing methods.By summarizing the reported nucleic acid probes,some limits and new challenges are gradually exposed.1)How to overcome the requirement of double labeled with fluorophore and quencher,or false positive responses due to dyes nonspecific adsorption to bimolecular;2)How to avoid the design of multiple nucleic acid probes and concomitant non-specific amplification in one sensing system;3)How to satisfy the analysis of trace biomolecules in complex systems,especially analysis at the single cell level;4)How to construct endogenous substance-actived logic platfomt in living cells,and then serve to accurate recognition of cancer cells with different phenotypes?In view of the above limitations and challenges,we have developed a series of novel functionalized nucleic acid probes for fluorescent assay of disease-related DNA fragments,DNA methyltransferase activity,mRNA in living cells and cancer cell recognition with different phenotypes.The main contents are as follows:Chapter one is an introduction section,and it summarizes the design principle and application of classic nucleic acids probes,and novel nucleic acid probes based on nucleic acid amplification and micro/nano materials.In addition,the existing limits and chanlleges of the present nucleic acid probes are also summarized.In chapter two,based on DNA templated Ag NCs(DNA/Ag NCs)fluorescent probe,a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1(HIV-1)DNA.Firstly,a hairpin probe,containing target DNA recognition sequence and guanine-rich sequence,was designed to hybridize with the target DNA and form a blunt 3'-terminus DNA duplex.Then,exonuclease ?(Exo ?)was employed to stepwise hydrolyze the mononucleotides from formed blunt 3'-terminus DNA duplex,releasing the target DNA and guanine-rich sequence.Then the released target DNA can hybridize with a second hairpin probe and initiate the next cycle,leading to more and more guanine-rich sequence.Finally,DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence,leading to an enhanced fluorescence signal for detection.The proposed method could detect as low as 2.9 × 10-10 mol L-1 HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA.Furthermore,the method possessed perfect recoveries in cells lysate and human serum,showing potential to be used in biological samples.In chapter three,based on target-protected dumbbell molecular probe(D-probe)mediated cascade rolling circle amplification(CRCA)strategy,an accurate and sensitive DNA methyltransferase(MTase)activity assay was developed.Firstly,D-probe serves as enzyme-linkage recognition probe of MTase and endonuclease.Next,methylated D-probe maintains its integrity and acts as the circular template for CRCA.Finally,mutiple D-probe copies of CRCA products bind with multimolecules labeling of Sybr Green I(SG),leading a cooperative amplified fluorecence signal for detection.Compared to the reported methods based on dsDNA or hairpin DNA molecular probes,the strategy is unique in characteristics:(?)D-probe here serves together as DNA MTase and endonuclease recognition probe,CRCA template,and signal probe,thus avoids the demand of multiple probes design.(?)Once unmethylated D-probe is cleaved into two parts by endonuclease,the CRCA could be effectively blocked,thus lowers the nonspecific amplification.(?)Cooperative amplification coupling with the CRCA and multimolecules labeling of SG can guarantee an improvement of sensitivity for MTase activity assay.(??)After the enzyme-linkage recognition of DNA MTase and endonuclease,the D-probe could be directly amplified by CRCA process.The more straight forward detection mode may achieve more simple and sensitive MTase activity assay.This work showed excellent specificity and sensitivity with a detection limit of 0.0024 U/mL,and potential application in quantitatively monitoring MTase activity and screening of anticancer drugs.In chapter four,based on a single integrated magnetic microprobe,we developed an ultrasensitive and accurate DNA(cytosine-5)-methyltransferase 1(Dnmt1)activity assay at the single-cell level.The single integrated magnetic microprobe is a bioconjugate of functionalized double-stranded DNA(dsDNA)anchored to a single magnetic microbead surface.Functionalized dsDNA is designed with a hemimethylated DNA site for Dnmtl recognition and a single-stranded tail to trigger in situ rolling circle amplification(RCA).Under the action of Dnmt1,hemimethylated dsDNA could be recognized and catalyzed to fully methylated dsDNA,which would protect them from the cleavage of BssHII.But the dsDNA without full methylation would be cut by BssHII,making single-stranded tail separated from the single integrated microprobe.Subsequently,full methylation-protected in situ RCA could be performed and multiple signal probes were hybridized to the single integrated microprobe for amplified signal accumulation.Finally,Dnmtl activity could be evaluated by reading the fluorescence of the single integrated microprobe.Meanwhile,to minimize matrix interferences,magnetic separation was performed in the process.In this strategy,the single integrated magnetic microprobe was provided with integrated capacities of target recognition,signal amplification,signal accumulation and matrix isolation.Therefore,an ultralow detection limit of 0.007 U/mL Dnmtl was obtained and accurate Dnmtl activity assays in multiple cell lysates at the single-cell level were achieved.Furthermore,the inhibition effect of RG108 was evaluated conveniently.These results indicate that the single integrated magnetic microprobe-based strategy is an excellent candidate for sensitive monitoring of Dnmtl activity and screening of anticancer drugs.In chapter five,based on programmable DNA-AuNPs spherical nucleic acid(SNA)probes,a intracellular logic platform was developed for mRNA imaging and recognition of cancer cells with different phenotypes.Epithelial-mesenchymal transition(EMT)is a phenomenon and process of adherent epithelial cells to a migratory mesenchymal state during tumor metastasis.At the molecular level,EMT involves the cellular loss of epithelial markers and increased expression of mesenchymal markers.Thus,EMT-related mRNAs have different expression levels in cells with different phenotypes and can be used to identify cells with different phenotypes.C-myc is an effective activator of tumorigenesis and related to the occurrence and development of various diseases.It is over-expressed in cancer cells(such as breast cancer),but not in normal cells,thus it can be used to recognize cancer cells from normal cells.In this work,we first constructed DNA-AuNPs SNA-based YES gates for the analysis the expression level of EMT-related mRNA and C-myc mRNA in breast cells with different phenotypes.Then,the DNA-AuNPs SNA-based AND gates was constructed for the simultaneous analysis of EMT-related mRNA and C-myc mRNA to identify breast cells with different phenotypes The design of intracellular mRNA-based DNA computation will show great applicaiton potential in mRNA imaging in CTCs,CTCs phenotypes recogniton and tumor heterogeneity analysis.Chapter six is a conclusion section,and it mainly summarizes the innovation of the paper.