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CN-121987782-A - Intelligent nano classifier based on glutathione gating as well as preparation method and application thereof

CN121987782ACN 121987782 ACN121987782 ACN 121987782ACN-121987782-A

Abstract

The invention discloses a glutathione-based gated intelligent nano classifier and a preparation method and application thereof, wherein the preparation method comprises the following steps of firstly, annealing four DNA chains T 1 、T 2 、T 3 、T 4 in an annealing buffer solution to form Programmable Atomic Nanoparticles (PAN); annealing the four DNA chains H 1 、H 2 、H 3 、H 4 in an annealing buffer solution to form four hairpins, and incubating the Programmable Atomic Nanoparticles (PAN) and the four hairpins to assemble the intelligent nano classifier. The intelligent nano classifier can be used for realizing the spatial selectivity visualization and the cooperative photodynamic therapy of various circrnas. The intelligent nano classifier can realize sensitive quantification of the circRNA under single-cell resolution, distinguish the circRNA from mismatched variants thereof on single-base resolution level, and can accurately identify breast cancer and lung cancer in different stages.

Inventors

  • ZHANG CHUNYANG
  • LIU QIAN
  • WANG LIJUAN
  • MA FEI

Assignees

  • 东南大学

Dates

Publication Date
20260508
Application Date
20260129

Claims (10)

  1. 1. A preparation method of an intelligent nano classifier based on glutathione gating is characterized by comprising the following steps: Annealing the four DNA chains T 1 、T 2 、T 3 、T 4 in an annealing buffer solution to form programmable atomic nano particles, and annealing the four DNA chains H 1 、H 2 、H 3 、H 4 in the annealing buffer solution to form four hairpins; Incubating the programmable atomic nanoparticles and the four hairpins to assemble the intelligent nano classifier.
  2. 2. The method according to claim 1, wherein in the first step, the single strands of DNA T 1 、T 2 、T 3 、T 4 、H 1 、H 2 、H 3 and H 4 are dissolved in 1 XTris-EDTA buffer, respectively, to prepare a stock solution, and diluted to 10. Mu.M with 1 Xannealing buffer, and the four strands of DNA T 1 、T 2 、T 3 、T 4 and the four strands of DNA H 1 、H 2 、H 3 、H 4 are heated at 95℃for 5 minutes, respectively, and then cooled slowly to 25 ℃; The 1 Xannealing buffer included 100 mM Tris-HCl,15 mM MgCl 2 , pH 8.0.
  3. 3. The method according to claim 1, wherein in the second step, the incubation is carried out at 37℃for 30 minutes.
  4. 4. The method according to claim 1, wherein the DNA strand T 1 has a sequence of 5'-GGT GGT GGT GGT TGT GGT GGT GGT GGG GAT GGG CAT GCT CGT GAC ATG ATC ATT AGT TTT T-3'; The sequence of the DNA strand T 2 is 5'-AAA GCC CGT GCG CTA ATG ATC ATG TCT CTG GAC CCT CGC ATG GAC GCT GTG AAC-3'; the sequence of the DNA strand T 3 is 5'-TTT TTC ATG CGA GGG TCC AGT CCA GCT TGC TAC ATT TTT-3'; The sequence of the DNA strand T 4 is 5'-GAG CAT CCA ATG TGT AGC AAG CTG GTC GAG CAT GCC CAT CCG AAA GCC CCG TG-3'; DNA strand H 1 sequence 5′-CGC ACG GGC TTT TTT TTT ACA ATC CTT TCA ACC TTT CCC ACA TTG GAT GCT CTG GGA AAG GTT GAA A/iHS-SH/GG ATT GT-3′; In the DNA strand H 1 ,/iHS-SH/represents disulfide bond modification; DNA strand H 2 sequence 5′-TTT CCC AGA GCA /iCy5dT/CC AAT GTG GGA AAG GTT GAA ACA TTG GA/iBHQ3dT/ GCT CTT TTT TGT TCA CAG CGT C-3′; In the DNA strand H 2 ,/iCy 5 dT/represents modification of a fluorophore Cy5,/iBHQ3 dT/represents modification of a quencher (BHQ 3) on the T base; DNA strand H 3 sequence 5′-CAT TGG ATG CTC TTT TTT TAG TAC CGA GAT TGT AGA TAT ATT CAA GTG TCG CTA TAT CTA CAA TCT C/iHS-SH/GG TAC TA-3′; In the DNA strand H 3 ,/iHS-SH/represents disulfide bond modification; DNA strand H 4 sequence 5′-AGA TAT AGC GAC AC/iCy3dT/ TGA ATA TAT CTA CAA TCT CAT TCA AG/iBHQ2dT/ GTC GCT TTT TTC ACG GGG CTT TC-3′; In the DNA strand H 4 ,/iCy dT/represents modification of a fluorophore Cy3 at the T base,/iBHQ dT/represents modification of a quencher (BHQ 2) at the T base.
  5. 5. A glutathione-based gated intelligent nanoscaler prepared by the method of claim 1.
  6. 6. The use of a glutathione-based intelligent nanoscaler of claim 5 for the preparation of a reagent for detecting circRNA.
  7. 7. The application of claim 6, wherein the intelligent nano classifier based on glutathione gating sensitively detects circCDYL and circHIPK3 and realizes accurate tumor treatment; the circCDYL sequence is 5'P-CCA UGG CCA CAG GCU UAG CUG UUA ACG GGA AAG GUU GAA AGG AUU GUU GAC AAA AGG AAA AAU AAA-3'; The circHIPK sequence is 5'P-UUU GUU CAA CAU AUC UAC AAU CUC GGU ACU ACA GGU AUG GCC UCA CAA GUC UUG GUC UA-3'; In circCDYL sequences and circHIPK sequences, "P" represents a phosphate group modification.
  8. 8. The method according to claim 7, wherein the detection is carried out by adding circCDYL and circHIPK3 to 10. Mu.L of the reaction solution followed by incubation at 37℃for 60 minutes; Wherein the reaction solution contains a concentration of 200 nM of glutathione based intelligent nano-classifier, 8 mM of glutathione, and 1x reaction buffer comprising 20 mM HEPES, 150 mM NaCl and 5mM MgCl 2 .
  9. 9. The method of claim 7 or 8, wherein the reagent has a minimum limit of detection circCDYL of 18.6: 18.6 aM and a minimum limit of detection circHIPK3 of 24.5: 24.5 aM.
  10. 10. The application of the intelligent nano classifier based on glutathione gating in the preparation of the medicine for photodynamic therapy of tumor, which is disclosed in claim 5, is characterized in that the medicine can induce apoptosis of tumor cells and can realize the apoptosis rate of 59.4% of cancer cells.

Description

Intelligent nano classifier based on glutathione gating as well as preparation method and application thereof Technical Field The invention belongs to the technical field of biological analysis, and particularly relates to a preparation method for exciting a glutathione-gated intelligent nano classifier, and application of the intelligent nano classifier in-situ imaging and accurate tumor treatment of various circrnas in living cells. Background Circular RNAs (circrnas) are a novel class of covalently closed non-coding RNA molecules. The pre-mRNA (pre-mRNA) is joined by a non-classical splicing mechanism of reverse splicing, a downstream 5 'splice donor site with an upstream 3' splice acceptor site, ultimately forming a circRNA with a circular structure. The circrnas have diverse biological functions including interacting with proteins, participating in protein translation, acting as a miRNA sponge to adsorb micrornas to regulate gene expression, and inhibiting host genes by competing with the classical splicing pathway. Abnormal changes in the expression level of circRNA are closely related to the occurrence and development of various diseases (such as cardiovascular diseases, nervous system diseases and neoplastic diseases) and cancers (such as gastric cancer, lung cancer, breast cancer, colorectal cancer, esophageal cancer, bladder cancer and osteosarcoma). Therefore, the circRNA has potential as a cancer biomarker, and has important significance for disease diagnosis and development of treatment schemes. Traditional methods for detecting circRNA mainly comprise Northern blot hybridization technology, circRNA sequencing technology (Circ-seq) and real-time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR). However, these in vitro detection methods require extraction of total RNA from tissue or cell samples and do not allow for imaging of the circRNA. To achieve real-time imaging analysis of endogenous circRNA, researchers have attempted to detect the level of circRNA in living cells using fluorescent DNA probes. However, this technique requires the use of transfection reagents to deliver the molecular beacon into the cell. In the subsequent research, scientific researchers further develop organic/inorganic nano materials as reaction probes and drug carriers, and although the cell uptake efficiency is improved, the high-concentration nano materials can cause cytotoxicity, and the accurate regulation and control of the modification process still have great challenges. In contrast, DNA nanostructures constructed based on watson-crick base pairing principle possess typical nanoscales, excellent biocompatibility, structural stability, precise molecular-level spatial arrangement ability, and excellent programmability. The characteristics can accurately regulate and control the distance, valence state and spatial distribution of molecules such as nucleic acid, polypeptide, protein and the like, and further, the method has good application prospects in various fields such as molecular imaging, drug delivery, tumor treatment and the like. The cross-shaped DNA nano structure provides an important thought for the development of the circRNA detection technology due to the advantages of strong mechanical rigidity, stable conformation, flexible design and the like. Furthermore, due to the heterogeneity of expression of circrnas, most existing detection methods for single circrnas often have difficulty in providing comprehensive and reliable detection information. In order to analyze multiple kinds of circrnas with different lengths and secondary structures in living cells, the problems of (1) reasonably designing target recognition modules to avoid cross interference among the circrnas in view of high sequence homology among the circrnas, (2) realizing synchronous cell delivery of multiple functional modules by means of efficient vectors, and (3) establishing a selective signal amplification strategy in view of extremely low expression abundance of the circrnas in the cells so as to improve detection accuracy are solved. Thus, constructing a multi-dimensional molecular classifier would provide a highly efficient tool for cell typing based on multiple circrnas. Nevertheless, the construction of a highly integrated molecular classifier to achieve synergistic integration of circRNA imaging with targeted therapies remains a challenge to be addressed. Photodynamic therapy (PDT) is the induction of oxidative damage and apoptosis of tumor cells by the production of Reactive Oxygen Species (ROS) and the consumption of oxygen (O 2) by photosensitizers under laser irradiation. The therapy has the advantages of minimally invasive property, rapid curative effect, difficult generation of drug resistance and the like. However, the hydrophobicity, targeting and sustained activation characteristics of the photosensitizer are insufficient, which often limit the application range of photodynamic therapy