Aptamer of methotrexate, aptamer derivative and application of aptamer and aptamer derivative

A technology of nucleic acid aptamer and methotrexate, applied in biological testing, biochemical equipment and methods, instruments, etc., can solve the problems of large toxic and side effects, narrow therapeutic window, endangering the life safety of patients, etc., and achieve small molecular weight, The effect of small batch variance and high stability

Active Publication Date: 2021-08-13
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, MTX has the problems of narrow therapeutic window and large toxic and side effects. The adverse reactions induced by high-dose methotrexate (HDMTX) mainly include nephrotoxicity, bone marrow suppression, mucositis, gastrointestinal reactions and neurotoxicity. May endanger the life of the patient

Method used

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  • Aptamer of methotrexate, aptamer derivative and application of aptamer and aptamer derivative
  • Aptamer of methotrexate, aptamer derivative and application of aptamer and aptamer derivative
  • Aptamer of methotrexate, aptamer derivative and application of aptamer and aptamer derivative

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Methotrexate nucleic acid aptamer screening.

[0037] 1. Synthesize a random single-stranded DNA library (GGAGGCTCTCGGGACGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTCGTCCCGATGCTGCAATCGTAA), a biotin-labeled complementary strand to the single-stranded DNA library (TCCCGAGAGCCTCCTTTT-Biotin), a forward primer (GGAGGCTCTCGGGACGAC) and a biotin-labeled reverse primer (GGCAGACGATT)

[0038] 2. Detailed screening process ( figure 1 )as follows:

[0039] Step 1: Take 1 nmol of ssDNA library strand and 5 nmol of complementary strand in 250 μL PBS (0.1M) buffer solution at 95°C for 5 min and then place at room temperature for 1 h;

[0040] Step 2: Take 200 μL streptavidin agarose balls to the micro-separation column, wash the streptavidin agarose balls in the separation column three times with PBS (200 μL / time), and then heat the denatured mixture Add to a miniature separation column and incubate with streptavidin agarose balls at room temperature for 10 min, collect the efflu...

Embodiment 2

[0053] Example 2: Construction of strand displacement biosensors.

[0054] The DNA sequence of FAM-HMX38 (FAM-CTCTCGGGCGAACGCGGGATGTTTGGGGGACCCACGTTCGCCC) tagged with FAM at the 5' end and cap38-BHQ1 (CGTTCGCCCGAGAG-BHQ1) tagged with BHQ1 at the 3' end was synthesized.

[0055] The synthesized DNA powders were all dissolved in PBS and quantified to 10 μM by Nanodrop. Take 10 μL of FAM-HMX38 (final concentration of 2 μM), 30 μL of cap38-BHQ1 (final concentration of 6 μM) and 10 μL of PBS, heat at 95 °C for 5 min, and then place at room temperature for 1 h. cap38-BHQ1 can complementarily pair with the 5' end of FAM-HMX38 to form strand displacement biosensor 38-3 (final concentration 2 μM, this concentration is based on FAM-HMX38), such as Figure 4 . Add different concentrations of MTX (0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM) to the above sensor 38-3 (final concentration 0.1 μM), react at room temperature for 30 min, and measure with a microplate reader. Its fluorescence v...

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Abstract

The invention discloses an aptamer of methotrexate, an aptamer derivative and application of the aptamer and the aptamer derivative. A nucleotide sequence of the aptamer comprises DNA molecules as shown in sequences 1-5. The aptamer can be a derivative obtained from various similar sequences with high homology or derivatives provided by the invention. A method for detecting the methotrexate is established by utilizing the specific binding effect of the aptamer HMX38 and the methotrexate for the first time. The aptamer and the derivative thereof can be used for preparing methotrexate detection probes and drug carriers or used for drug design and development, drug separation and purification and the like. The aptamer and the derivative thereof have the advantages of being capable of being combined with methotrexate with high specificity and high affinity, free of immunogenicity, capable of being chemically synthesized, good in biocompatibility, small in molecular weight, stable, easy to store and the like.

Description

technical field [0001] The present invention relates to a nucleic acid aptamer, nucleic acid aptamer derivatives and applications thereof, in particular to a nucleic acid aptamer capable of binding methotrexate, a nucleic acid aptamer derivative and its ability to recognize and bind methotrexate Application of pterin. Background technique [0002] Methotrexate (MTX), a folic acid antagonist, is widely used in the clinical treatment of various cancers and inflammatory diseases. According to reports, nearly 700 kinds of cancer cells are sensitive to MTX, among which acute lymphoblastic leukemia cell lines and acute myeloid leukemia cell lines are the most sensitive to MTX. MTX can interfere with the synthesis of tetrahydrofolate, thereby inhibiting the synthesis of DNA and RNA in cells, thus playing an anti-tumor effect. However, MTX has the problems of narrow therapeutic window and large toxic and side effects. The adverse reactions induced by high-dose methotrexate (HDMTX)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115G01N33/5308C12N2310/16
Inventor 施国跃何俊清汪俊彦张闽
Owner EAST CHINA NORMAL UNIV
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