Novel Theranostic Strategies Mediated By In Cellulo Catalytic Hairpin Assembly Circuits

Studying the expression level of important biomolecules can help us under stand the nature of life,the pathogenesis and development of diseases and find appropriate treatment strategies.In recent years,the development of early diagnostic methods for major diseases has attracted increasing interest of many researchers.Nucleic acid probes have become an excellent tool for studying some important biomarkers in life processes due to their ease of synthesis,good biocompatibility,easy functionalization and the specific bases pairing.On the basis of various existing nucleic acid signal amplification technologies,we developed new methods for highly sensitive imaging of two tumor markers(microRNA-21 and telomerase)in cancer cells and corresponding imaging guidance therapy by combining a nucleic acid molecular isothermal signal amplification technology,Catalytic Hairpin Assembly(CHA)with antisense nucleic acid technology.The main research contents and results are as follows:(1)Constructing nucleic acid probe for microRNA detection and theranostics.The expression level of several intracellular miRNAs is closely related to the occurrence of tumors.Establishing effective methods to detect intracellular miRNAs is helpful for early diagnosis of tumors and providing appropriate treatment options to prolong patients' survival.However,due to their low expression level,detection of intracellular miRNA is still a great challenge.In Chapter 2,we designed a group of nucleic acid probes by combining antisense gene therapy technology with traditional CHA technology and realized the integration of diagnosis and treatment.We chose streptavidin(SA)as a vector due to its good biocompatibility and constructed two kinds of DNA tetrads through the specific binding between biotin labeled on the end of DNA strands and SA.The tetrads can enter cells through a unique endocytosis mechanism.Under the trigger of intracellular miRNA-21,the hybridization reaction between probes on the two tetrads can be triggered and release fluorescence resonance energy transfer(FRET)signal.At the same time,the hybridization products expose the antisense sequence which is hidden in the two substrates sequence,enabling knocking out of downstream Bcl-2 mRNA by RNase H.The methods we developed in this chapter have the potential to be applied to the detection o f other cancer-related genes in cells and the knockout of downstream genes for cancer treatment.(2)Developing nucleic acid probe for in situ sensitive telomerase detection.Telomerase is a ribonucleoprotein reverse transcriptase composed of a catalytic subunit and the template RNA of telomere reverse transcriptase.It is a common and reliable tumor marker.In Chapter 3,we developed a new method for detecting intracellular telomerase based on the characteristics of telomerase that can recognize and prolong its substrate sequence.We designed and optimized two hairpin sequences H1 and H2 as CHA substrates and used telomerase elongated products as the trigger chain.After transfecting the substrate strands and the prime sequence into cells,the intracellular telomerase will bind to the prime sequence and extend the sequence to form the initiator of CHA,which then triggers hybridization between the two substrates H1 and H2.This restores the fluorescence of fluorophore groups labeled on H1 hairpin which used to be close to each other with quenched fluorescence.This strategy demonstrated high sensitivity for the detection of telomerase.Cell imaging showed that it can be applied to the detection of telomerase in different cell lines.