Construction Of Activated Aptamer Functionalized DNA Nanoprobes And Their Appliction For Tumor Extracellular ATP Imaging

As an important small molecule with biological activity in organism,ATP participates in various physiological activities,dysregulation of ATP homeostasis is closely related to occurrence and development of a variety of diseases.Therefore,it is of great significance to develop specific detection and imaging methods for ATP.Traditional ATP detection methods,such as electrochemical and high performance liquid chromatography are often used for the detection of ATP in solution,while optical analysis based on molecular probe has its advantages in the analysis of ATP at cellular level.Aptamer provide a tool for the construction of molecular probes with specific target recognition ability.At the same time,with the development of DNA nanotechnology,a variety of DNA nanostructures provide a carrier platform for the construction of novel molecular probes.Thus,in this paper,we use aptamer as the targeted recognition molecule and combine DNA nanotechnology to carry out the construction of activated aptamer functionalized DNA probe and the research of tumor extracellular ATP imaging.Specifically,it includes the following two aspects:1.Construction of split activated aptamer functionalized DNA triangular prism probe and tumor extracellular ATP imagingBased on the highly specific target recognition function of aptamer,we used Sgc8 c Aptamer,which specifically recognizes PTK-7 protein,and split ATP Aptamer,which specifically recognizes ATP,as the targeted recognition molecule,combining the signal readout mode based on fluorescence resonance energy transfer(FRET)"OFF" to "ON" and the signal activation mode based on the split probe assemble after recognition,successfully constructed an activated DNA triangular prism probe for tumor extracellular ATP imaging.In this probe,two fragments of split ATP Aptamer were complementary paired with the 3’ and 5’ ends of one edge of the DNA triangular prism,respectively,and each ends were labeled with Cy3 or Cy5 fluorescence groups,respectively.Sgc8 c Aptamer binds to the base of the DNA triangular prism and promotes the binding force of the probe to tumor cells by targeting PTK-7 protein.In the absence of ATP,the two fluorescence groups move away from each other,and FRET is in the "OFF" state,where the Cy5 fluorescence signal is barely visible at 561 nm excitation light.In the presence of high concentrations of ATP,split ATP Aptamer binds to ATP to form a terpolymer complex that drives the Cy3 and Cy5 at both ends to move closer to each other,FRET is then in the "ON" state,and there is visible Cy5 fluorescence signal at 561 nm excitation light.The results showed that the probe has high sequence specificity,good selectivity,low detection limit and the ATP imaging ability at the cellular level.2.Construction of activated aptamer functionalized DNA probes that recognition-triggering configuration changes and tumor extracellular ATP imagingIn view of the limitations of the DNA triangular prism probe in the previous chapter,such as complex sequence construction of three-dimensional DNA nanostructures,easily endocytosis,weak binding force of split ATP Aptamer,and limited recovery of FRET signal,in this chapter,we design a two-dimensional aptamer functionalized DNA probe(AFDP)for tumor extracellular ATP imaging by combining the more intuitive signal readout mode based on "OFF" to "ON" of a single fluorophore group and the signal activation mode based on the probe recognition-triggering configuration changes.The probe contains three sequences,one of which contains complete ATP Aptamer and marks BHQ1 quenched group in the middle,and can form hairpin structure itself(S-ATP);One sequence(S-Sgc8)containing Sgc8 c Aptamer and labeled FAM fluorescence group at the 5 ’end;There is also a bridge sequence named(S1).The three chains were annealed to form an activation probe.In the presence of a single activation unit(PTK-7 protein or ATP),the separation of S-ATP and S-Sgc8 could not be induced.the fluorescence group FAM and quenched group BHQ1 remained close to each other,and the fluorescence signal was in the state of "OFF".When the probe targets to tumor cells,it would cause conformational changes of Sgc8 c Aptamer,resulting in the release of the bridge chain S1,and the S1 opens the hairpin structure and exposes the functional domain of ATP Aptamer.in the tumor microenvironment with high ATP concentration,ATP Aptamer conformation changes and separates with S-Sgc8.FAM and BHQ1 are further away to produce "ON" fluorescence signal.Experimental results show that the probe has dual target activation ability,more sensitive ATP response ability and more intuitive imaging results.The design of the probe provides a new idea for tumor extracellular ATP imaging.