The Research Of New Methods For Early Detection Of Tumors Based On Strand Displacement Probes

Molecular imaging and in vitro molecular diagnosis are two important techniques for micro-or non-invasive detection of early tumors.Unlike classical imaging,molecular imaging,such as fluorescent molecular imaging,can detect abnormalities at the cellular and molecular levels during the early formation of tumors.However,the development of molecular imaging technology is still in the preclinical experimental stage and is far from mature,and the targeting or specificity of most molecular imaging probes needs to be improved.On the other hand,circulating tumor DNA(ctDNA)has become one of the hotspots of in vitro molecular diagnostic research because of its rich tumor-related genetic information or mutation information.With the help of PCR technology and DNA hybridization technology,some ctDNA detection reagents have been used in the diagnosis of clinical tumor diseases and have benefited many patients.Because the content of ctDNA in the specimen is very small,and the low-abundance of ctDNA is often accompanied by a large number of wild-type genes,which has high requirements for the sensitivity of ctDNA detection technology.However,the sensitivity of the existing ctDNA detection technology is limited,and tumor samples with corresponding low-abundance mutations(<1%)ctDNA will be misdiagnosed as negative.Both molecular imaging and molecular diagnostics need to develop molecular probes with high specificity and sensitivity.This research focuses on the current problems in the clinical application of molecular imaging and molecular diagnostics,and further explore the specificity of DNA strand replacement probes and their possibilities in clinical applications.Here are some brief descriptions for the research content.In Chapter 2,we established a tetrahedron DNA nanostructure and gold nanoparticle-based nucleic acid nanosensors for the detection of miRNA-21 in living cells.We specially designed the bases of the toehold part of the probe to control the Gibbs free energy.Make the Gibbs free energy change of the strand displacement reaction close to zero.Under this condition,small energy differences caused by single base mismatches can cause significant changes in hybridization efficiency.Gold nanoparticles were used to load specially designed molecular probes,and DNA tetrahedrons(TDN)were used to co-transport molecular probes to construct nano-sensors(Au-TDNNs).Then,based on fluorescence resonance energy transfer,a method for miRNA-21 fast detection in living cells was developed.The rationally designed TDN sequence and the probe sequence do not interfere with each other,and therefore,the nano-sensors can be easily and quickly assembled.We tested single-base mismatched miRNA-21 analogs,let-7d and miRNA-200 b,and the best single base mismatch discrimination factor(DF)for the specially designed molecular probes is 15.4.As a control group,the DF of ordinary molecular probes reached only 2.4.The use of liver cancer cells(HepG2)further validates the feasibility of this sensing strategy.The coordinated transport of Au-NPs and TDN allows the optimal imaging time in the cell to be advanced to 1.5 hours.Therefore,this study provides an effective method for rapid and specific in situ imaging of small molecules in living cells.In Chapter 3,we used the Bulge-loop structure to regulate the specificity of the probe and obtained highly specific probes.In the previous chapter,we adjusted the base composition of the toehold part of the strand displacement probe to improve the specificity of the probe.However,due to the limited degree of change in the base sequence of the toehold,it is still a challenge to adjust the thermodynamic equilibrium of the strand displacement reaction.In this chapter,we adjust the recognition ability of SNV by introducing the Bulge-loop structure into the strand displacement probe.Based on the controllable regulation of the free energy change(?G)by the Bulge-loop,the probe obtained much higher specificity than the conventional linear strand displacement probe.In order to reduce the impact of wild-type genes on point mutation detection,we have designed competitive shadow probes to block wild-type genes.The kinetic test results confirmed that the detection limit of the probe to the L858 R mutation can reach 0.02%.With the introduction of the Bulge-loop structure,we obtained a more specific strand displacement probe.In Chapter 4,we used the Bulge-loop probe to build a nucleic acid sensor to detect low-abundance point mutations in tumor samples.Many molecular detection methods perform well in the detection of synthetic DNA samples,but cannot be used directly in the detection of clinical samples of tumors.PCR is the most important amplification technology for molecular diagnostics.We compared the effects of two PCR technologies by using Bulge-loop probe.We found that the method of ? exonuclease hydrolysis of PCR products is more conducive to the detection of low-abundance point mutations,referred to here as enzyme digestion method.Then we used a standard addition method to prepare a series of L858 R mutation samples in 10% serum.The Bulge-loop probe combined with enzyme digestion method obtained a sensitivity of 0.1%.More importantly,the Bulge-loop probe correctly identified low-abundance L858 R mutant lung cancer samples that could not be detected by commercial PCR kits or Sanger sequencing.This result is highly consistent with the droplet digital PCR(ddPCR)result.This study provides a new method for establishing highly sensitive molecular diagnostic techniques.