DNA Logic Nanodevices For Spatially Selective Imaging Of Tumor Cells

Malignant tumors pose a great threat to human life owing to difficulty in early screening,low cure rate,and easy recurrence.Highly expressed biomolecules in cancers are available to specifically distinguish tumor cells from normal cells for early diagnosis of tumors.In addition,a large number of signaling molecules are released during the development and treatment of cancers to indicate tumor progression and treatment effects.Therefore,the early screening for malignant tumors and signaling molecules are conducive to the realization of early diagnosis of tumor cells,monitoring of the development process,and real-time formulation of personalized tumor treatment solutions.In medical research,imaging techniques have been developed to obtain information of cancers,such as X-ray tomography,magnetic resonance imaging,and ultrasound imaging,which cannot determine the multi-molecular information and monitor the physiological and pathological processes in real-time.Fluorescence molecular imaging can simultaneously monitor multiple targets to obtain molecular information of tumor cells in real-time.Therefore,it is necessary to construct molecular fluorescence imaging probes with simple preparation,good biocompatibility,high sensitivity and accuracy.There are many problems such as single target,low detection sensitivity and unreliably information in the existing fluorescent imaging probes of tumor cells.DNA nanoprobes have been widely used in the imaging of tumor cells owing to good biocompatibility,high programmability,and controllable modification.In particular,DNA logic nanodevices have attracted widespread attention with multiple inputs,logical processing of information,and high accuracy.However,there are still three problems in the existing research:(1)Low detection sensitivity and insufficient information accuracy are caused by the single level and dimension of inputs.(2)Diffuse components of DNA logic nanodevices resulted in slow logic operations.(3)Studies with poor spatial selectivity cannot monitor physiopathological processes in specific regions of tumor cells.Based on this,this thesis aimed to improve the performance of DNA logic nanodevices,such as sensitivity,accuracy,operation speed and spatial selectivity by preparing three new DNA logic nanodevices for spatially selective imaging of tumor cells.It included the following three aspects:1.DNA logic nanodevices with confinement-effect for sequential imaging of transmembrane cancer markers.Due to the discovered biomarkers with different spatial distributions in tumor cells,monitoring different spatially distributed cancer biomarkers in living cells is critical to obtain precise and multidimensional information on cell types and cancer progression.In this study,DNA logic nanodevices integrated confinement effect and AND gate logic operation into DNA tetrahedron were constructed for transmembrane sequential imaging Mucins 1(MUC1)on cell membrane and micro RNA-21(miR-21)in cytoplasm to achieve accurate identification of tumor cells.When MUC1 was expressed on the cell membrane surface,MUC1-apt fell off to release Cy5 signals and exposed the toehold of H1 for binding to miR-21,realizing MUC1 as a recognition switching of miR-21 and avoiding circuit leakage as the results shown.After the nanodevices entered cells,intracellular miR-21 initiated the catalytic hairpin assembly(CHA)reaction between H1 and H2 to output Cy3 signals.Sequential logic response to MUC1 and miR-21 significantly improved the detection accuracy.Due to the spatial confinement effect of DNA tetrahedron,the reaction speed and efficiency between H1 and H2 were significantly improved to detect miR-21 sensitively and rapidly.This study enabled accurate,rapid,and sensitive imaging of multidimensional biomarkers for precise differentiation of tumor cell types.2.Smart DNA logic nanodevices for dynamic imaging of H~+/ATP in lysosomes.In the first work,DNA logic nanodevices suffered from low loading for cell imaging.Moreover,challenges still remained in constructing DNA nanodevices to perform logic operations at the organelle level.Based on this,the acid-responsive i-motif sequence(T1),ATP aptamer(T2)and SH-modified linker strand(L)were hybridized to form Y-shaped DNA structures(Y-DNA),which then were grafted on gold nanoparticles(AuNPs)by Au-S bonds to construct DNA logic nanodevices for selective imaging H~+/ATP in lysosomes.As results shown,T1 formed an i-motif structure in the acidic medium and high concentrations of ATP specifically bond to T2 to output fluorescence resonance energy transfer(FRET)signals between Cy3 and Cy5 for dynamic imaging of H~+/ATP in lysosomes.In this study,multiple energy transfer and AND gate logic operations were integrated into AuNPs to form DNA logic nanodevices,reducing false positive signal leakage and improving logic operation speed of multi-target detection.The proposed DNA logic nanodevices were expected to verify the immune-enhancing effect of autophagy activators in the treatment of immunogenic cell death and the diagnosis of lysosomal-related cellular diseases.3.Multifunctional DNA logic nanodevices for real-time monitoring mitochondrial micro RNAs during cell apoptosis.In the second work,DNA logic nanodevices had low detection sensitivity for molecules in subcellular organelles.In addition,few studies have monitored multiple micro RNAs(miRNAs)in mitochondria during cell apoptosis.Based on this,DNA nanospheres were used as carriers to extend two backbone chains to efficiently encode H1 and H2 which can cycle to multiple targets.Triphenylphosphine was modified in the skeleton of DNA nanospheres to achieve mitochondria-targeting function.Thereby,DNA logic nanodevices integrated multi-target cycling and mitochondrial-targeting functions were constructed.Only when the tumor cells expressed simultaneously miR-21 and miR-10b,CHA reaction between H1 and H2 was initiated to output energy transfer signals from Cy3 to Cy5 and realize the simultaneous cyclic amplification for two targets.The speed and efficiency of logic operations were significantly increased owing to spatial confinement effect of DNA nanospheres,improving the sensitivity of the CHA reaction to miR-21 and miR-10b.The proposed nanodevices can be selectively localized in the mitochondria with the help of triphenylphosphine to monitor the changes of mitochondrial miR-21 and miR-10b levels in real-time,sensitively and rapidly during cell apoptosis.In summary,DNA logic nanodevices were constructed by mainly utilizing the programmability of DNA nanostructures and multi-level inputs for spatially selective imaging of tumor cells to obtain precise and multi-dimensional information of tumor cells.Therefore,the following problems have been solved.Firstly,DNA logic nanodevices were constructed by integrating signal amplification and multi-level logic inputs into DNA tetrahedron for transmembrane sequential imaging of MUC1 and miR-21 to accurate diagnosis of cancer cells.To improve the loading efficiency,acid responsive i-motif and ATP aptamers were efficiently modified on the surface of AuNPs to construct DNA logic nanodevices for the dynamic monitoring of H~+/ATP in lysosomes,achieving DNA logic operations at the organelle level.To further realize spatially selective multi-target sensitive imaging,the hairpins for CHA reaction were efficiently encoded on DNA nanospheres to construct logic nanodevices with simultaneous amplification for miR-21 and miR-10b to improve the sensitivity and speed of logic operation.The logic nanodevices were selectively localized in mitochondria to monitor miR-21 and miR-10b in real-time during cell apoptosis,which was of great significance for the study of cell apoptosis mechanism and the optimization of cancer treatment solution.