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Nucleic acid aptamer capable of simultaneously recognizing multiple malignant tumor cells and application of nucleic acid aptamer

A nucleic acid aptamer and malignant tumor technology, applied in the field of molecular diagnosis and treatment, to achieve the effect of small molecular weight, non-immunogenicity and good reproducibility

Active Publication Date: 2022-05-17
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, at present, there is no report of nucleic acid aptamers specifically targeting undifferentiated thyroid cancer cells at home and abroad, and this application fills this gap

Method used

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  • Nucleic acid aptamer capable of simultaneously recognizing multiple malignant tumor cells and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of simultaneously recognizing multiple malignant tumor cells and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of simultaneously recognizing multiple malignant tumor cells and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Screening of nucleic acid aptamers capable of simultaneously recognizing human undifferentiated thyroid cancer cells and human papillary thyroid cancer cells

[0038] 1. Design of the nucleic acid library and primers used:

[0039] Random Nucleic Acid Library (SEQ ID NO: 1):

[0040] 5'-ATCCAGAGTGACGCAGCA(N 44 )TGGACACGGTGGCTTAGT-3'

[0041] Upstream primer (SEQ ID NO:2): 5'-fluorescein isothiocyanate-ATCCAGAGTGACGCAGCA-3'

[0042] Downstream primer (SEQ ID NO: 3): 5'-biotin-ACTAAGCCACCGTGTCCA-3'. Wherein, N represents any random base of A, T, C, and G.

[0043] The sequence of the nucleic acid aptamer PT-2 is as follows (SEQ ID NO: 4):

[0044] 5'-ATCCAGAGTGACGCAGCACTGTGGCGTTACTTGAGTCCCTTC C TACAAGCCCTGGATTAGAGTGTGGACACGGTGGCTTAGT-3', wherein the replacement of the 43rd base "C" with "T" does not affect the secondary structure and binding properties of the sequence, and the sequence list after replacement is shown in SEQ ID NO:5.

[0045] 2. The speci...

Embodiment 2

[0076] Example 2: Identify the target type of nucleic acid aptamer PT-2, and determine its binding affinity to target cells

[0077] 1. Preliminary identification of PT-2 target types

[0078] Before incubating with PT-2, CAL-62 was first digested with trypsin and proteinase K to treat the cells, the purpose is to destroy the cell membrane protein, if the target of PT-2 binding is membrane protein, the cells after proteinase K treatment no longer PT-2 binding. Such as Figure 6 As shown, the digestion of trypsin can significantly weaken the combination of PT-2 and CAL-62, and the CAL-62 cells treated with proteinase K no longer bind to PT-2. Therefore, it is preliminarily speculated that the target of PT-2 is membrane protein.

[0079] 2. Determination of PT-2 binding dissociation constant

[0080] The Cy5-labeled aptamer PT-2 was prepared at final concentrations of 500nM, 400nM, 300nM, 250nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.563nM, 0.781nM, 0.391nM, 0.19...

Embodiment 3

[0081] Example 3: Observing the effects of different incubation temperatures on the binding of PT-2 to target cells

[0082] The Cy5-labeled nucleic acid aptamer PT-2 was prepared into a binding solution with a final concentration of 250 nM, and the target cells were inoculated into a glass bottom confocal culture dish with a diameter of 35 mm 24 hours in advance. Incubate with target cells for 1 hour at 4°C and 37°C respectively, then wash three times with 4°C washing buffer, and observe the subcellular localization of fluorescent signals under a confocal microscope. Such as Figure 8A As shown in , B, C and D, it was found that PT-2 was localized on the surface of CAL-62 cells and TPC-1 cell membranes at 4°C; at 37°C, PT-2 entered the CAL-62 cell membrane and localized in the cytoplasm. For TPC-1 cells, PT-2 was localized on the cell surface at 4°C, while at 37°C it was localized around the nuclear membrane and entered into the nucleus.

[0083] Similarly, the Cy5-labeled...

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Abstract

The invention discloses a nucleic acid aptamer capable of simultaneously recognizing multiple malignant tumor cells and application of the nucleic acid aptamer. The nucleic acid aptamer PT-2 is screened from human undifferentiated thyroid cancer CAL-62 and papillary thyroid adenocarcinoma TPC-1, and the nucleic acid sequence of the PT-2 is shown as SEQ ID NO: 4 in a sequence table. The nucleic acid aptamer is obtained by screening living cells, not only can be combined with human undifferentiated thyroid carcinoma and papillary thyroid carcinoma cell lines in a high-affinity manner, but also can be combined with various other tumor cell lines with higher malignancy degrees in a high-affinity manner; the nucleic acid aptamer PT-2 is free of immunogenicity, small in molecular weight, easy to modify, replace and mark and capable of being chemically synthesized in vitro; meanwhile, the aptamer is good in sequence stability, easy to store and convenient to mark; the aptamer synthesis cost is lower than that of the antibody, the period is short, and the reproducibility is good; the invention can be applied to detection of human undifferentiated thyroid carcinoma, papillary thyroid carcinoma and related malignant tumor cells, and can be used for preparing related therapeutic drugs.

Description

technical field [0001] The invention relates to a nucleic acid aptamer for simultaneously recognizing multiple malignant tumor cells and its application, belonging to the field of molecular diagnosis and treatment. Background technique [0002] Malignant tumors are one of the most serious diseases that threaten human health in modern society. It poses a great threat to human health and life, and brings a heavy economic burden to the sick family and the whole society. The World Health Organization ranks the treatment of malignant tumors as the primary task of human health. Taking thyroid cancer as an example, thyroid cancer (thyroid carcinoma) is derived from thyroid malignant tumors, accounting for about 1% of systemic malignant tumors. Papillary thyroid carcinoma is a differentiated thyroid cancer derived from thyroid follicular epithelial cells. It is the most common pathological type of malignant thyroid tumors. The incidence of thyroid cancer is more than 80%. Undiffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/569G01N33/574
CPCC12N15/115G01N33/56966G01N33/574C12N2310/16
Inventor 蒲颖印明柱陈翔
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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