Study On PiRNA Imaging And Cancer Theranostic Based On Multi-Functionalized DNA Tetrahedron Probes

As a novel cancer biomarker,PIWI-interacting RNA(piRNA)is closely related to the occurrence and development of cancer.Therefore,it is of great importance to the development the precise,safe and efficient cancer-related piRNAs involved theranostic probes for determining cancer progression and realizing accurate theranostics of cancer.At present,the scientific reports focus on the detection methods of piRNAs,such as Northern Blot,PCR,next-generation RNA sequencing(RNAseq)analysis and nanomaterial-based probe methods.Among them,the nanomaterial-based probe methods can achieve non-invasive,real-time and in situ detection of piRNAs.However,the cancer-related piRNAs involved theranostic strategies are rarely reported.Nanoprobes,which have small size,h igh affinity and excellent stability,are widely used in the detection of cancer biomarkers.Meanwhile,nanoprobes have the advantages of flexible design and convenient modification,so that they can accommodate multiple functional elements and evolve the simplex piRNAs detection probes into the multifunctional theranostic nanoprobes.Nevertheless,with the development of multifunctional integrated theranostic nanoprobes,their safety and biocompatibility have bec ome non-negligible factors in the design strategies of nanoprobes.The appearance of DNA nanostructures provides a new perspective to solve this problem.DNA nanostructures are nucleic acid nanostructures which formed by self-assembly of oligonucleotide chains with the Watson-Crick base complementary pairing principle.As its biosafety and biocompatibility,DNA nanostructures have been widely used in the biomedical field.In addition,DNA nanostructures own natural structural rigidity and unlimited design ceiling,that can integrate multiple function al elements while maintaining stability and providing a powerful tool for the development of cancer theranostic nanoprobes.In this dissertation,selected cancer-related piRNAs as the targets,three kinds of cancer-related piRNAs involved theranostic nanoprobes based on DNA nanostructures were constructed,through introducing homogeneity-owned functional nucleic acid molecules such as Aptamer and antisense oligonucleotide(ASO),embedding chemical anticancer drug Doxorubicin(DOX)in C-G of DNA double-stranded structure,resulting in the accurate theranostics for the breast cancer as model.The detailed description is listed as follows:1.Aptamer-functionalized activatable DNA tetrahedron nanoprobe for PIWI-interacting RNA imaging and regulating in cancer c ellsIn order to construct a theranostic cancer nanoprobe that can respond cancer-related piRNA,piRNA-36026 is selected as a proof of concept for the study in this chapter,while the assembly stands for DNA tetr ahedron are functionalized as follows: Aptamer AS1411 sequence is extended from the assembly stands S1 and S2,and the antisense sequence of piRNA-36026 is extended from the other two assembly stands S3 and S4 with Cy5 fluorophore modified at the extension end.The assembly strand Q-oligo is a sealing strand with BHQ2 at the end,which can partially complement the piRNA-36026 antisense sequence in S3 and S4.The five strands are self-assembled to form aptamer-functionalized activatable DNA tetrahedron nanoprobes(apt-ADTNs).The apt-ADTNs are able to rapidly recognize the target MCF-7 cells and enter the cells by the Aptamer AS1411,then,the over-expressed piRNA-36026 in the cytoplasm compete with the S3 and S4 to supplant the Q-oligo,resulting in the recovery of Cy5 fluorescence to realize activated imaging of piRNA-36026.Meanwhile,the combination of piRNA-36026 with its antisense sequence down-regulates the expression of free piRNA-36026 in the cytoplasm,which interferes with the signaling pathways of piRNA-36026 involved in cancer molecules,leading to the apoptosis of MCF-7 cells.The results show that apt-ADTNS can simultaneously realize imaging and regulation of piRNA-36026 in MCF-7 cells,which provides a new idea for the design of piRNAs invo lved nanoprobes for cancer theranostics.2.Activable dual cancer-related RNAs imaging and combined gene-chemotherapy through the target induced intracellular disassembly of functionalized DNA tetrahedronThe work in the previous chapter demonstrated that the f unctionalized DNA tetrahedron nanoprobes can realize the specific fluorescence imaging and regulation of piRNA-36026 in cancer cells.However,the accuracy of cancer diagnosis by imaging of the single cancer-related RNA is insufficient,and the killing eff ect through regulating the only one RNA is not significant.Therefore,we select two breast cancer-related RNAs(piRNA-36026 and m RNA Bcl-2)as targets,combine with two cancer treatment modalities(gene therapy and chemotherapy),and use the "assembly and disassembly" strategy to develop an activat able Two & Two type of cancer theranostic system(AT&TCTS)for the diagnosis and therapy of breast cancer.The AT&TCTS is based on the DNA tetrahedron structure and further functionalized as follows: the antisense sequence of Bcl-2 m RNA is designed in S1 with modification of Cy3.The Aptamer AS1411 sequence is extended from S4,and S5 is designed to contain the antisense sequence of piRNA-36026 with modification of Cy5.The BHQ2 s are respectively modified at the corresponding position of S2 and S3,which can quench the fluorescence of Cy3 and Cy5.After the five strands are assembled into the functional DNA tetrahedron skeleton,the chemotherapeutic drug DOX is embedded into the functional DNA tetrahedron skeleton through the C-G base of the DNA double strand to form AT&TCTS.AT&TCTS targets MCF-7 cells through its Aptamer AS1411 and then enters them.At this time,piRNA-36026 and m RNA Bcl-2overexpressed in the cytoplasm respectively bind to the corresponding antis ense sequences in AT&TCTS,activate fluoresc ence signals of Cy3 and Cy5 to image the dual targets,respectively.This process can down-regulate intracellular dissociative m RNA Bcl-2 and piRNA-36026,hinder their involved molecular signal pathways and lead to apoptosis of MCF-7 cells.Meanwhile,this process of the combination between targets with AT&TCTS leads to the disassembly of AT&TCTS and the release of DOX embedded in AT&TCTS to achieve the chemotherapy for cancer cells.The results of this study confirm that AT&TCTS is able to image accurately and treat effectively for breast cancer at the cellular and in vivo levels,which provides a new perspective for the development of multiple cancer related RNAs involved cancer theranostic system with high precision and effective efficacy.3.Logic operation for accurate identification of breast cancer utilizing dual PIWI-interacting RNAs recognizing functionalized-DNA tetrahedron theranostic system with combined gene-chemotherapyThe activatable Two & Two type of cancer theranostic system constructed in t he previous chapter "assembly-disassembly" strategy is an independent activation system of two targets in cancer cells,which may have the problem of insufficient accuracy in judging cancer categories.In order to further improve the accuracy of cancer diagnosis and ensure the therapeutic effect,we choose two overexpressed piRNAs(piRNA-651 and piRNA-823)as targets to develop a cancer theranostic system,which is base on the structure of DNA tetrahedron,introdu ces nucleic acid logic gate strategy,combine gene-chemotherapy treatment mode,for the accurate identification and the combination therapy of gene-chemotherapy for breast cancer.Firstly,the assembly stands for DNA tetrahedron are functionalized.S1 and S2 are designed with the piRNA-651 antisense sequence(anti-piR-651)and modified with Cy5 fluorophore.S3 and S4 are extended with Aptamer AS1411 sequence.Yb is designed as a sealed sequence modified with BHQ2.Yc is containing the antisense sequence of piRNA-823(anti-piR-823)and partially complementary antisense sequence of piRNA-651 and Yb.The framework of the functionalized cancer theranostic system is assembled through the six strands,and then the chemotherapeutic drug doxorubicin(DOX)is loaded into the C-G base of DNA double helix in the functionalized DNA tetrahedron framework to form the functionalized-DNA tetrahedron cancer theranostic system with logically responding for dual piRNAs(Dp FDTCTS).The aptamer on it enables Dp FDTCTS to recognize and enter target MCF-7 cells.In the cytoplasm,the overexpressed piRNA-651 and piRNA-823 can response Dp FDTCTS logically.In brief,piRNA-823 can bind with anti-piR-823 in Yc,separates Yc from Dp FDTCTS and exposes the anti-piR-651 in S1 and S2.Then,piRNA-651 binds with anti-piR-651,separates Yb from Dp FDTCTS and activate Cy5 fluorescence to image MCF-7 cells.This process of logical response of piRNA-823 and piRNA-651 to Dp FDTCTS down-regulate the expression level of the two piRNAs,leading to the gen e therapy for MCF-7.Meanwhile,this process partially disassembled Dp FDTCTS,resulting in partially releasing of DOX which shows the chemotherapy effect for MCF-7 cells.The experimental results indicate that Dp FDTCTS performs specifically logic response to piRNA-823 and piRNA-651 at the cellular and in vivo levels,which realizes specific fluorescence imaging of breast cancer and increases the accuracy of diagnosis for breast cancer.In addition,combination therapy significantly improves the treatment efficiency of breast cancer.It provides a new method for the precise theranostic of breast cancer.