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Aptazyme sequence

A nucleic acid aptamer and ribozyme technology, applied in biochemical equipment and methods, instruments, analytical materials, etc., to achieve the effects of high sensitivity, improved sensitivity and low cost

Inactive Publication Date: 2019-03-08
HUNAN INSTITUTE OF ENGINEERING
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, there is currently no highly sensitive and specific ATP detection method based on nucleic acid aptamers, ribozymes and hybridization chain reactions. The ATP detection sensitivity of existing methods can only reach the nanomolar level, and the detection of ATP has been It is widely used in many aspects including tumor drug sensitivity screening, cell apoptosis detection, various important cell activity and microbial content analysis, etc. With the deepening of research and the development of trace analysis technology, scientists are more sensitive to ATP There is a strong demand for detection, so the development of a more sensitive ATP detection method is of great significance for medical clinical diagnosis and body metabolism research.

Method used

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Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1 ATP nucleic acid aptamer ribozyme probe design

[0051] (1) The structure of the ATP nucleic acid aptamer ribozyme probe is as follows:

[0052]

[0053] The first line "CATCTCTTC" and the second line "GAAGAGATG" (attached figure 1 Parts A and A' in the middle) complementarily hybridize to form the stem in the molecule; the second line "TACTTAAACA" and "TGTTTAAGTA" part (attached figure 1 The H and H' parts in the middle) complementarily hybridize to form the stem-loop G in the molecule; the first line "AGCGATCTA" (SEQ ID NO.1) is the 10-23 ribozyme sequence (attached figure 1 Middle part B), the first line "GGGGGAGTATTGCGGAGGA" (SEQ ID NO.2) part is the ATP nucleic acid aptamer sequence (attached figure 1 F in part), the second line "AGAAGAAGGTGTTTAAGTA" (SEQ ID NO.3) part (attached figure 1 Middle G and H' parts) are the priming sequences of the hybridization chain reaction. When there is no ATP, this sequence is closed in the stem of the molec...

Embodiment 2

[0060] Embodiment 2 is to the detection of different concentration ATP

[0061] (1) Formation of ATP aptamer ribozyme secondary structure

[0062] The biotin-modified aptamer ribozyme uses 25mM HEPES buffer (25mM HEPES, 100mM NaCl20mM MgCl 2 ,pH=7.4) configuration. In order to reduce the generation of non-hairpin dimers and increase the formation ratio of hairpin structures, stepwise slow annealing was adopted. Take 100 μL of 1 μM biotinylated aptamer ribozyme in an EP tube, and gradually anneal as follows: 95°C for 5 minutes, 60°C for 5 minutes, 40°C for 5 minutes, 30°C for 10 minutes, 20°C for 15 minutes, and store at 4°C.

[0063] (2) Formation of secondary structures of hairpin probes P1 and P2

[0064] Hairpin probes P1 and P2 use 25mM HEPES buffer (25mM HEPES, 100mM NaCl 20mM MgCl 2 ,pH=7.4) configuration. Take 100 μL of 0.5 μM hairpin probes P1 and P2 in EP tubes, and gradually anneal slowly in order to reduce the generation of non-hairpin dimers and increase the for...

Embodiment 3

[0070] The specificity of embodiment 3 ATP detection

[0071] In order to test the specificity of the sensing system for ATP detection, three other interfering molecules (UTP, GTP, CTP) were selected and their interference degree to ATP detection was tested respectively. The concentration of the four small molecule compounds is 1nmol / L. From the results, the detection signal of ATP is much greater than that of other interfering molecules. See the attached Figure 5 , which shows that the technical system has high specificity to ATP and can meet industrial clinical application.

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Abstract

The invention discloses a high-sensitivity detecting probe based on an aptazyme sequence and an intermolecular splitting G-quadruplex-hemin DNAses self-assembled nanowire, a kit and a preparation method and application thereof, and is particularly suitable for detection of adenosine triphosphate (ATP). The invention combines an aptamer, Mg<2+>-dependent 10-23 ribozyme, hybridization chain type reaction and the G-quadruplex-hemin DNAses enzymatic reaction, thereby realizing stepwise amplification of a detecting signal and substantially improving the detecting sensitivity which is up to the pmol / L level and is higher than the general detecting sensitivity level nmol / L of the prior art by 3 magnitudes. An ATP aptazyme probe is excellent in working performance, has the detecting sensitivity limited to 2 pmol / L, and is extremely high in specificity. Common UTP, GTP, CTP and other interfering substances have no effect on ATP detection; requirements of medical clinical diagnosis, metabolic studies and the like for an ultrahigh-sensitivity ATP detecting method can be met; and the high-sensitivity detecting probe is extremely high in popularization potential and actual application value inthe industry.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-sensitivity detection probe, a kit and the The preparation method and application can be used for the detection of adenosine triphosphate (ATP). Background technique [0002] Adenosine triphosphate (ATP), a high-energy phosphate compound, is considered a universal energy source essential in the cellular synthesis of all living organisms to survive and reproduce, and figuratively speaking, it is a universal energy "currency". Since Lohmann et al. first discovered and extracted ATP from muscle with strong sugar catabolism in 1929, it has been proved to play an important role in the metabolism of fat, protein, sugar and nucleic acid in living cells and in maintaining the normal function of organisms. important role. By monitoring the change of ATP content, the cell killing, cell inhibition and cell proliferation caused by various drugs, biological agents or biological active su...

Claims

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Application Information

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IPC IPC(8): C12N15/11G01N33/573C12Q1/682
CPCG01N33/573C12Q1/682G01N2333/9005
Inventor 张何李雯黄幸芳傅昕杨梅
Owner HUNAN INSTITUTE OF ENGINEERING
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