Development Of Several Functional Molecule Detection Methods Based On DNA Nanotechnology In Cells

When the content of functional molecules in the cell,telomerase,miRNA,ATP,and methylated DNA changes,it is crucial for the occurrence and development of cancer.Therefore,the quantitative detection of the content of intracellular functional molecules is of great significance for the early diagnosis of cancer and the evaluation of drug efficacy in cancer treatment.Highly sensitive quantitative detection of functional units in cells faces many challenges.First,the content of many functional groups in cells is shallow to achieve the purpose of early diagnosis,it requires some effective signal amplification methods and efficient detection methods;then,because many diseases are not a single marker can be accurately diagnosed,so a variety of functional molecules in a unique cell series of quantitative detection of the design of the detection probe and the accuracy of the detection method has higher requirements;finally,because of the particular location of methylated DNA in the cell,the expansion of the detection probe requires the size of the probe to allow it to be in the cytoplasm and nucleus free shuttle,to achieve better detection and quantitative results.This paper studies and demonstrates the above problems.Through the efficient,accurate and stable combination of nanomaterials and DNA nanotechnology,we have successfully proposed the quantitative detection method of intracellular telomerase;the simultaneous quantitative detection strategy of telomerase,miRNA and ATP;and the simultaneous quantitative detection approach of telomerase and methylated DNA.The above programs provides a reliable basis for the early diagnosis and treatment of cancer.The main contents are as follows:1.Highly Sensitive Detection of Telomerase Activity in CellsAs an important biomarker for early cancer diagnosis and a valuable therapeutic target,the detection and monitoring of telomerase activity has attracted extensive attention.Herein,we constructed a novel fluorescent reporter to detect the intracellular telomerase activity based on guanine enhanced sliver nanoclusters(AgNCs).In this system,a telomerase substrate(Ts)was designed to be extended by telomerase to give the repeat TTAGGG sequence as G-rich DNA,which was complementary to the nine base sequence to form a self-hairpin structure.The proximity of G-rich DNA to the as-prepared AgNCs resulted in a 13-fold enhancement of fluorescence intensity due to the electron transfer from guanine to the AgNCs.Therefore,the fluorescent probe could realize the detection of telomerase activity.Furthermore,the probe was successfully used to distinguish normal cells from cancer cells and to monitor the real time telomerase activity response upon treatment with an inhibiting model drug.This detection technique is simple,with excellent biocompatibility,inexpensive and expands the applications of the AgNC probe.2.Simultaneous Quantitative Detection of Telomerase,Mi RNA and ATP in CellsBecause of insufficient information,a single biomarker is not sufficient for early diagnosis of cancer,whereas sensitive and selective detection of multiple biomolecules can significantly reduce analysis time,sample size,and accurately perform cell screening in early cancer.Therefore,the development of a noninvasive strategy that can simultaneously quantify multiple biomarkers(i.e.,nucleic acids,proteins,and small molecules)in a single cell is particularly important.Herein,a universal sensing system(functional DNA@mesoporous silica nanoparticles(MSN)-Black Hole Quencher-rhodamine 6G(Rh B),FDSBR),which is based on the combination of functionalized DNA and smart responsive nanomaterial,was successfully constructed.After incubation with the cells,different types of targets trigger the strand displacement reaction to release the fluorophore-labeled nucleic acids as the output signals to reflect the intracellular level of the telomerase and adenosine triphosphate(ATP),respectively.Simultaneously,intracellular miR-21 can be clearly indicated by the restored fluorescence of Rh B when the caged double-stranded DNA was substituted into single-stranded DNA to open the pore.The concentrations of intracellular telomerase,miR-21,and ATP were identified successfully in three cell lines at the single-cell level.The results show that the contents of three biomolecules have significant differences in the three model cell lines and provide a promising route for developing innovative early disease diagnosis and cell screening assay.3.Simultaneous Quantitative Detection of Telomerase and DNA Methylation in CellsTelomerase plays an important role in many biological processes.DNA methylation regulates the expression of many genes including telomerase.However,a strategy for simultaneously monitoring the activity of telomerase and DNA methylation in a cell has not yet been constructed.Here,we propose a deformable nano-satellite nanocapsule(FITC-hollow bowl mesoporous organicsilica@gold nanoparticles-Methyl-Cp G-binding protein 2(MECP 2)-silver nanoclusters,FHBMO@AMA),simultaneous quantitative detection of the cytoplasmic telomerase activity and the DNA methylation level.This strategy enables a spatial-based detection in cells.The total cytoplasmic telomerase activity was detected by fluorescence energy resonance transfer(FRET)between FHBMO and gold nanoparticles(Au NPs).And the DNA methylation in the nucleus was detected by enhanced fluorescence of silver nanoclusters(AgNCs).Furthermore,FHBMO@AMA can also intuitively distinguish the difference in telomerase expression in cells in S/M phases.Interestingly,the ratio of the two detections(telomerase activity/DNA methylation)is significantly correlated with the efficacy of the anticancer drugs,while there is no obvious linear relationship between the any single detection target and the efficacy of the anticancer drugs.Therefore,based on the relationship between telomerase activity and DNA methylation,it could develop a new and feasible method for evaluating the efficacy of anticancer drugs,which can extend the technology toolbox for precision medical and pharmaceutical analysis.