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Composition and method for regulating any gene expression

A technology of gene expression and gene regulation, applied in the field of molecular biology, to achieve the effect of wide applicability, strong versatility, and beneficial to in vivo delivery

Pending Publication Date: 2021-11-12
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, so far, there is no gene regulation tool that can act on DNA targets and RNA targets at the same time; it can work in prokaryotic cells and eukaryotic cells; Up-regulate gene expression and down-regulate gene expression

Method used

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  • Composition and method for regulating any gene expression
  • Composition and method for regulating any gene expression
  • Composition and method for regulating any gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1

[0046] In order to verify whether the AfuFEN1 protein (the nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2) can successfully target and bind the target substrate ssDNA under the guidance of the mis-hpDNA probe, Such as figure 2As shown, we designed several probes with a stem-loop structure at the 3' end of these probes, and the 5' end was basically complementary to the target ssDNA, but did not match the target ssDNA at some specific positions. The expressed and purified AfuFEN1 protein was incubated with these probes at 37°C for 20 min, and then ssDNA was added to incubate for 30 min, and the reaction products were analyzed by EMSA.

[0047] Such as figure 2 As shown, when there is no mismatched base on the probe (PC group, hp4-17), the substrate in the swimming lane is almost completely cut (marked as cleavage product); when the mismatched base on the probe is in position 1 (hp4-17-m1), there i...

Embodiment 2

[0049] Example 2

[0050] In order to investigate whether AfuFEN1 protein can fully bind the target substrate ssDNA under the guidance of mis-hpDNA, such as image 3 As shown, we gradually increased the amount of AfuFEN1 protein. As the ratio of FEN1 protein to the target ssDNA continued to increase, the amount of the "ssDNA-probe-FEN1" ternary complex gradually increased, indicating that AfuFEN1 was involved in mis-hpDNA probes. Guided by (hp4-17-m), it can better bind to the target substrate (ssDNA-S2).

[0051] In order to further improve the binding efficiency, we increased the complexity of the stem-loop structure at the 3' end of the mis-hpDNA probe, respectively version 1.0 (hp4-19-m-ver1.0), 1.1 (hp4-19-m- ver1.1), 1.2 (hp4-19-m-ver1.2) and 1.3 (hp4-19-m-ver1.3) probes. Such as Figure 4 As shown, as the complexity of the stem-loop structure increases, the amount of the "ssDNA-probe-FEN1" ternary complex also gradually increases. The above results indicated that A...

Embodiment 3

[0054] Example 3

[0055] In order to investigate whether AfuFEN1 can bind target substrate RNA under the guidance of mis-hpDNA probe, such as Figure 5 As shown, we expressed and purified AfuFEN1 protein with 1.0 (hp4-19-m-ver1.0), 1.1 (hp4-19-m-ver1.1), 1.2 (hp4-19-m-ver1.2 ) and version 1.3 (hp4-19-m-ver1.3) of the mis-hpDNA probe were incubated at 37°C for 20 minutes, and then the target substrate ssRNA (ssRNA-S2) was added for incubation for 30 minutes, and the reaction products were analyzed by EMSA. The ternary complex composed of "ssRNA-probe-FEN1 protein" marked by the dotted line box can be observed; this result shows that AfuFEN1 can efficiently bind the RNA target substrate under the guidance of the mis-hpDNA probe.

[0056]

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Abstract

The invention discloses a composition and a method for regulating any gene expression. The composition comprises an oligonucleotide probe and a protein molecule; the oligonucleotide probe is composed of two parts, one part of the oligonucleotide probe is provided with a nucleic acid secondary structure, the other part of the oligonucleotide probe is basically complementary to a target substrate, and basic groups at certain specific positions are not complementary to the target substrate; the protein molecule can recognize the nucleic acid secondary structure of the oligonucleotide probe, so that the protein molecule is bound with the probe, is guided by the probe and is bound with the target substrate to form a 'target substrate-protein molecule-probe' ternary complex; based on the fact that the basic groups on the certain specific positions of the oligonucleotide probe are not complementary with the target substrate, the protein molecule only has a binding reaction with the target substrate, and a cleavage reaction does not occur. The composition not only acts on a DNA target, but also acts on an RNA target; the composition not only can work in prokaryotic cells, but also can work in eukaryotic cells; and the composition not only can up-regulate gene expression, but also can down-regulate gene expression.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a composition and a method for regulation and control of arbitrary gene expression. Background technique [0002] With the completion of the Human Genome Project, biological research has gradually entered the post-genome era. An important research theme in the post-genomic era is to explore gene functions and find regulatory networks; while the systematic characterization of gene functions and regulatory networks relies on the precise manipulation of genes through deletion, suppression or overexpression. Therefore, DNA and RNA manipulation tools, such as gene editing tools and gene regulation tools, can further advance the development of related research. [0003] Gene editing tools represented by ZFNs, TALENs, and CRISPR systems can cut DNA strands to insert deletions in the coding region of the target gene, causing a frame shift in the reading frame, and then destroyin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22
CPCC12N15/113C12N9/22C12N2310/531
Inventor 徐澍汪亮郭永健
Owner CHINA PHARM UNIV
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