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Nucleic acid polypeptide complex probe and preparation method and application thereof

A technology for polypeptide complexes and nucleic acids, which is used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of insufficient sensitivity for detection, etc., and achieves a simple, easy, and time-consuming preparation method. , highly selective effect

Pending Publication Date: 2019-07-19
NANJING MEDICAL UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

For low-abundance miRNAs, the sensitivity of our currently developed quasi-targeted proteomic assay may not be sufficient for detection, especially if the method is to be used in clinical practice

Method used

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  • Nucleic acid polypeptide complex probe and preparation method and application thereof
  • Nucleic acid polypeptide complex probe and preparation method and application thereof
  • Nucleic acid polypeptide complex probe and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Preparation and Identification of Example 1 Nucleic Acid Polypeptide Complex Probe

[0034] (1) Preparation and purification of nucleic acid-polypeptide complex probes

[0035] Using trichloroethyl phosphate as the reducing agent, 200 μL of 3’-end disulfide bond-modified DNA at a concentration of 1 μM: 5’-biotin-TCCATCATTACCCGGCAGTATTA-3’ (Sangon Biotechnology Co., Ltd., Shanghai) and 20 μL of TCEP reduction beads (Thermo Fisher Scientific, USA) mixed, reacted and shaken at 37°C for 2h; then centrifuged the sample at 1000×g for 6min; took the supernatant containing the reduced DNA prepared above, and added the same A volume of 20 μM N-terminal maleimide-modified substrate polypeptide: GDKAVLGVDPFR (Genetide Biotechnology Co., Ltd., Shanghai), shaking at 37°C for 4 hours for conjugation reaction, followed by using the difference in retention time before and after conjugation (DNA And the retention time of the DNA-polypeptide complex is 10.4min and 18.2min respectively, ...

Embodiment 2

[0038] Example 2 Double-strand-specific nuclease-mediated isothermal amplification strategy

[0039] (1) Optimization of conditions for double-strand-specific nuclease-mediated amplification

[0040] Double-strand-specific nuclease-mediated amplification relies on the specific enzymatic selectivity of double-strand-specific nucleases for double-stranded DNA or DNA strands in DNA:RNA hybrid duplexes. In this study, the occurrence of DNA-polypeptide complex and miR-200c hybridization and double-strand-specific nuclease cleavage was confirmed by HPLC analysis. Such as Figure 6 As shown, the retention times of miR-200c and nucleic acid-polypeptide complex probes were 7.3 and 17.9 minutes, respectively. After hybridization with excess miR-200c, the nucleic acid-polypeptide complex probe peak disappeared, and a newly formed hybridization product appeared at 17.3 minutes. After further treatment with double-strand-specific nuclease, only partial DNA-polypeptide fragments generate...

Embodiment 3

[0053] Example 3 Quantification of miR-200c in BCSCs using double-strand-specific nuclease-mediated amplification combined with LC-MS / MS

[0054] Using the method in Example 2, the content of miR-200c in MCF-7 cells and breast cancer stem cells was measured, which were (5.86±1.40)×10 3 copies / cell and (3.80±0.66)×10 2 copies / cell. The level of miR-200c in breast cancer stem cells was lower than that in cancer cells (p Figure 11 ). Compared with our method, the level of miR-200c detected by qRT-PCR was slightly lower, but no significant difference was observed between the two methods.

[0055] In addition, we quantified miR-200c in magnetically sorted stem cells from 16 human breast cancer tissue samples. The results showed that BCSCs accounted for 12.1 ± 4.3% (range: 3.0-21.5%) of the total cells in the tumor ( Figure 12 ); the level of miR-200c was quantified as (1.43±0.96)×10 3 copy / breast cancer stem cell (range: (0.50-3.13)×103 copy / breast cancer stem cell) ( Figur...

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Abstract

The invention relates to a nucleic acid polypeptide complex probe and a preparation method and an application thereof. According to the invention, a structure is shown in the specification: the R1 isDNA: 5'-biotin-TCCATCATTACCCGGCAGTATTA-3'; and R2 is a substrate polypeptide: GDKAVLGVDPFR. The method combines a double-chain specific nuclease-mediated isothermal amplification strategy with a targeted proteomic technology for the first time, selectively degrades a DNA chain in a DNA chain of a DNA-polypeptide: miRNA hybrid by the double-chain specific nuclease, combines the cycle amplificationability to release a large number of nucleic acid polypeptide complex probe fragments with a very small amount of miRNAs with the high sensitivity, high selectivity and wide dynamic range of the polypeptide targeting a proteomics detection report, and develops a novel quasi-targeted proteomics method to quantifying low-abundance miRNAs, such as miRNAs in stem cells.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, and in particular relates to a nucleic acid-polypeptide complex probe and its preparation method and application. Background technique [0002] MicroRNAs (miRNAs) are a class of single-stranded non-coding RNAs with a length of 18-25 nucleotides. They play important regulatory roles in many biological processes by inhibiting the translation of messenger RNA (mRNA). Recent studies have found that the abnormal expression of miRNA is closely related to a series of cancers including breast cancer. Tumors are composed of heterogeneous cells, among which stem cells have the characteristics of self-renewal and multilineage differentiation ability, which are the most important reasons for tumorigenesis, maintenance, metastasis and recurrence. Therefore, it is reasonable to speculate that miRNAs are associated with key properties of cancer stem cells (CSCs). Moreover, increasing evidence in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2521/301
Inventor 陈芸匡雨琼许飞飞曹建翔王钟城
Owner NANJING MEDICAL UNIV
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