A kind of fluorescent probe and its application in detecting sirt2 enzyme activity

A fluorescent probe and probe technology, applied in fluorescence/phosphorescence, measurement device, material analysis by optical means, etc., to achieve the effect of low cost, simple operation and reduced error

Active Publication Date: 2020-12-01
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no relevant literature patent report that can continuously and not be coupled with any enzymatic reaction or chemical reaction to specifically detect the method of SIRT2 enzyme activity

Method used

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  • A kind of fluorescent probe and its application in detecting sirt2 enzyme activity
  • A kind of fluorescent probe and its application in detecting sirt2 enzyme activity
  • A kind of fluorescent probe and its application in detecting sirt2 enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Verify that fluorescent probes are not affected by SIRT2 or NAD + The effect of acting alone, including the following steps:

[0037] (1) Configure the following four groups of samples

[0038] Sample 1: Add 1 μl NAD to 47 μl buffer solution (50 mM HEPES; 100 mM KCl; 0.001% Tween-20; 0.05 mg / ml BSA; 200 mM TCEP; pH: 7.4, the same below) + (50mM, the same below), 1μl fluorescent probe (1mM, the same below), and finally add 1ul SIRT2 (15μM, the same below);

[0039] Sample 2: Add 1 μl fluorescent probe to 48 μl buffer, and finally add 1ul sirt2;

[0040] Sample 3: Add 1 μl NAD to 48 μl buffer + , and finally add 1ul of sirt2;

[0041] Sample 4: Add 1 μl fluorescent probe and 1 μl NAD to 48 μl buffer + ;

[0042] (2) The four groups of samples were all reacted at 37° C. for one hour, and after the reaction, the fluorescence spectrum from 380 nM to 580 nM was detected with a Flex Station3 multifunctional microplate reader under excitation of 320 nM.

[0043] Depend o...

Embodiment 2

[0045] The detection of the sensitivity of the fluorescent probe to the SIRT2 enzyme includes the following steps:

[0046] Add 1 μl fluorescent probe (1 mM) and 1 μl NAD to 47 μl buffer + (50mM), and finally add different concentrations of 1μl SIRT2 (final concentrations are 43.75nM, 87.5nM, 175nM, 350nM, 425nM, 500nM), using FlexStation3 multifunctional microplate reader at 37°C, excitation wavelength 320nM, emission wavelength 416nM continuous Detect for 30 minutes, and count the rate of fluorescence change within 30 minutes.

[0047] Depend on image 3 It can be seen that under the condition that the concentration of the fluorescent substrate remains unchanged, as the concentration of SIRT2 protein increases, the fluorescence enhancement rate of the system also increases.

Embodiment 3

[0049] The detection of the specificity of the fluorescent probe to the SIRT2 enzyme includes the following steps:

[0050] (1) Configure the following three groups of samples

[0051] Sample 1: Add 1 μl SIRT1 (15 μM), 1 μl NAD+, 1 μl fluorescent probe to 47 μl buffer.

[0052] Sample 2: Add 1 μl SIRT2 (15 μM), 1 μl NAD+, 1 μl fluorescent probe to 47 μl buffer.

[0053] Sample 3: Add 1 μl SIRT3 (15 μM), 1 μl NAD+, 1 μl fluorescent probe to 47 μl buffer.

[0054] (2) The three groups of samples were all reacted at 37°C, and the Flex Station3 multifunctional microplate reader was used to continuously detect the excitation wavelength of 320nM and the emission wavelength of 416nM for 30min, and the fluorescence change rate within 30min was counted.

[0055] Depend on Figure 4 It can be seen that at the same concentration, the sample group added with SIRT1 protein and SIRT3 protein basically has no fluorescence signal, while the SIRT2 sample group has a significant increase in ...

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Abstract

The invention belongs to the field of biological analysis and detection and discloses a fluorescent probe and the application thereof in detecting SIRT2 enzyme activity. The structure of the probe is:Ac-Gly-R1-R2-Thr-NH2 with R1 being a long chain acylated lysine of a fluorescent group and R2 being a quenching group. The probe of the invention is used for detecting SIRT2 enzyme activity, and thereaction system is not coupled with any enzymatic reaction or chemical reaction. According to the invention, the fluorescent group on the lysine side chain can be separated from the quencher group onthe substrate backbone only by one step that SIRT2 deacylates the substrate lysine residue. The activity of SIRT2 is then determined by detecting the fluorescence change of the fluorescent group at acertain excitation wavelength. The method provided by the invention has the advantages of simple operation, low cost, and is suitable for high-throughput screening. The method reduces the errors thatmay be introduced into the detection system by introducing other substances or other reactions, and enables continuous detection of SIRT2 activity and screening of SIRT2 inhibitors.

Description

technical field [0001] The invention belongs to the field of biological analysis and detection, and relates to a fluorescent probe and a method for detecting SIRT2 enzyme activity. Background technique [0002] Histone deacetylation modification plays an important role in gene expression and regulation. Sirtuin is a highly conserved catalytic reaction dependent on NAD+ deacetylases from bacteria to humans. The human Sirtuin family has seven members: SIRT1-SIRT7. They interact with p53, Ku70, FOXO, PGC-1α and other proteins in the human body, and participate in the regulation of cellular stress response, metabolism, aging and apoptosis. [0003] SIRT2 mainly exists in the cytoplasm, has strong histone deacetylase activity, and can deacetylate α-tubulin at lysine 40 both in vivo and in vitro. In addition, studies have found that SIRT2 can effectively remove long-chain acyl groups on lysine in vitro, which provides a new idea for the development of SIRT2 detection methods in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K5/103G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 刘培庆李民戴琪
Owner SUN YAT SEN UNIV
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