System Of Nucleic Acid Hybridization-based Tumor Molecular Diagnosis

Tumors are the leading cause of death in humans.The occurrence of tumors is mostly related to gene mutations,and 95%of them are caused by single base mutations.Meanwhile studies have shown that telomerase-mediated indefinite elongation of telomeres is also a key cause of tumorigenesis.Single base mutations and telomerase activity have been identified as biomarkers for tumor diagnosis.However,there are no obvious symptoms in the early stage of tumor development,which makes the clinical diagnosis difficult.When they are discovered,most are in the late stage and the cure rate is extremely low.Therefore,the detection of tumor-related molecules plays an important role in the early diagnosis and treatment of tumors.In this study,three novel detection methods of nucleic acid and protein conformation for highly selective detection of tumor-associated enzyme activity and single base mutation were established by means of nucleic acid stochastic binding,cationic conjugated polymer FRET and distance-dependent graphene induced energy transfer.First of all,because the elongation of telomeres has been associated with tumorigenesis,it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis.Currently,amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo.However,amplification is typically associated with poor reproducibility and high background,which hamper further applications of the strategies,particularly for real sample assays.Here,we establish a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy.The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a completely different kinetic signature from the background noise,allowing us to identify telomerase reaction products(TRPs)without background at the single molecule level.This background-free assay allows a limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection.And in the actual sample measurement,telomerase extracted from cancer cells was determined with sensitivity down to 10 cells.Moreover,the length distribution of TRPs was also determined for the first time by multiple stochastic probing,which could provide deep insight into the mechanistic study of telomerase catalysis.Next,DNA Hybridization-based probes emerge as a promising tool for nucleic acid target detection and imaging.However,the single-nucleotide selectivity is poor.Therefore,we develop an effective and simple method for highly selective detection and in situ imaging of single-nucleotide mutation(SNM)by taking the advantages of the specific hybridization of short duplex and the signal amplifying effect of cationic conjugated polymer(CCP)found in the previous part of the study.Excellent discrimination of the nucleic acid strands only differing by single nucleotide was achieved enabling the sensitive detection of SNM at the abundance(proportion of mutant gene in the sample)as low as 0.1%.Single-molecule fluorescence resonance energy transfer(smFRET)study reveals that the presence of CCP enhances the perfect matched duplex and the mismatched duplex to a different extent,which can be an explanation for the high single-nucleotide selectivity.Meanwhile CCP can also protect DNA probe from nuclease degradation.Due to the simple design of the probe and the stable brightness of CCP,highly selective mRNA in situ imaging was achieved in fixed cells.Cell imaging study reveals that melanoma cell line A3 75 with BRAF V600E point mutation exhibits higher FRET efficiency than liver cancer cell line HepG2 that was not reported having the mutation at this point.This method is a new approach for using hybridization-based DNA probe.It would find broad applications in DNA based sensing and intracellular imaging.To the end,detailed structural information of protein is the fundament for understanding its function,which is of key relevance for disease diagnosis and drug development.To date,atomic structures of numerous proteins have been resolved by X-ray crystallography and cryo-electron microscopy(cryo-EM).However,these powerful structural biology techniques require sophisticated and expensive instrumentation,complex sample preparation and data analysis,and are difficult to use for a large number of samples.In this work,we establish a "nanoruler" by using different lengths of DNA strands as fluorescent carriers,fixing the DNA strands to the graphene surface to obtain different distances between the fluorescent molecules and the graphene surface,and then quantifying the graphene fluorescence quenching ratio.To immobilize protein on graphene,functional graphene-supported lipid monolayer was established using trisNTA as the protein capturing moiety.Apply this ruler to the detection of axial alignment information of protein complexes.It was successfully detected that the Ypt7 protein undergoes a 2.1 nm conformational change under the influence of Mon1-Ccz1.For the first time,the relationship between the efficiency and distance of graphene fluorescence quenching was quantified experimentally.And the "nanoruler" can be applied to the detection of structural changes of various biomacromolecules.In summary,the three novel detection methods of tumor molecular diagnosis all have the advantages of simple operation,rapid detection,low cost,high specificity and high sensitivity.And there is the good potential of clinical application.