Development Of New Methods For Analysis Of Disease Markers Base On DNA Nanomachines

Diagnosis and screening of diseases is an important step before treatment.Simple and efficient detection methods can help doctors judge the condition and provide helpful propose for the treatment of diseases.Disease markers(such as certain nucleic acids,glycoproteins,enzymes,etc.)are closely related to certain diseases.They appear with or even earlier occurrence than diseases,reflecting the severity of diseases and the state of related cells.In this paper,two new detection methods for disease markers,miR-21 and UDG enzyme,were proposed.Methodology 1:To construct a diagnostic and therapeutic integrated DNA nano-machine with nano-golden ball as carrier to respond to the expression of microRNA-21 and Bcl-2 in cancer cells,and to simultaneously perform fluorescence imaging and drug treatment of microRNA-21 in cancer cells.Methods 2.Using the characteristics of UDG enzyme specific cleavage,we designed a switch to catalyze hairpin self-assembly reaction,and used DNA-stabilized silver nanoclusters as fluorescent signals to detect UDG enzyme activity.The main research contents are as follows:1.By modifying DNA chains with different functions on gold nanospheres,a diagnostic and therapeutic integrated DNA nanomachine with complex nanostructures is constructed.The specific DNA strands are used to activate the nano-machine in response to the double targets of miR-21 and bcl-2,which are highly expressed in cancer cells.Under the activated state,the specific DNA strands are based on the principle of chain replacement reaction.Antisense nucleic acid drugs carrying fluorescent groups on the "capture and release" machine are recycled to the surrounding environment,so that the fluorescent groups are far away from the nano-golden sphere and get rid of the FRET effect to generate fluorescence.At the same time,antisense nucleic acid knocks down the expression of Bcl-2 gene,which causes cell lysis and death,and realizes the integration of diagnosis and treatment.2.Based on the characteristics of UDG enzyme specific cleavage of DNA strands,a self-assembly reaction system of catalytic hairpin was constructed.The activity of UDG enzyme was specifically detected by fluorescence signal generated by DNA-stabilized silver nanoclusters.Firstly,DNA strand hairpin structures HP 1 and HP 2 with catalytic hairpin self-assembly ability and silver cluster stabilization sites were designed.Secondly,the barrier chain B containing a certain mismatched base U was designed to combine with the function of trigger chain I by the principle of base complementary pairing and to form a reaction system for detecting UDG enzyme.In the presence of UDG enzyme,chain B is blocked from being specifically cleaved and activated to trigger chain I to produce catalytic hairpin self-assembly cycle reaction,forming a large number of HP-1/HP-2 hybrid double-stranded structures.The signal output of DNA stabilization nanosilver cluster in HP-1 is detected by "lighting" the fluorescence of the G-enhanced functional region of HP-2.