KR20130069924A - Dna oligomer that binds as an antisense to the cyclin dependent kinase1 mrna - Google Patents

Dna oligomer that binds as an antisense to the cyclin dependent kinase1 mrna Download PDF

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Publication number
KR20130069924A
KR20130069924A KR1020110136975A KR20110136975A KR20130069924A KR 20130069924 A KR20130069924 A KR 20130069924A KR 1020110136975 A KR1020110136975 A KR 1020110136975A KR 20110136975 A KR20110136975 A KR 20110136975A KR 20130069924 A KR20130069924 A KR 20130069924A
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South Korea
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dna
complementary
sequence
cyclin dependent
mrna
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KR1020110136975A
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Korean (ko)
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김승찬
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김승찬
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Priority to KR1020110136975A priority Critical patent/KR20130069924A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR

Abstract

PURPOSE: A DNA oligomer is provided to enhance knock-down effect by interfering a stable DNA, to suppress various cancers, and to control cell proliferation and division by regulating cyclin dependent kinase 1 expression level. CONSTITUTION: A method for reducing cyclin dependent kinase 1 expression level in cultured cells or tissues and organisms or application thereof comprises a step of preparing a DNA oligomer with 10 or more base pairs, which complementarily binds to cyclin dependent kinase 1 mRNA. The DNA oligomer contains 3'-caaactttgacgagcgtg-5'(DNA strand which is complementary to upstream), 3'-catttaatattttaacat-5' (DNA strand which is complementary to midstream sequence), and 3'-cagaaggtgtgtatgaaa-5'(DNA strand which is complementary to downstream sequence). The DNA oligomer is prepared by synthesis of a PCR primer. [Reference numerals] (AA) Front sequence(underline = complementary strand, 3'---5' = antisense DNA sequence); (BB) Middle sequence(underline = complementary strand, 3'---5' = antisense DNA sequence); (CC) Rear sequence(underline = complementary strand, 3'---5' = antisense DNA sequence)

Description

DNA oligomer that binds as an antisense to the cyclin dependent kinase1 mRNA} which binds complementarily to mNNA for ecｙｃｌｉｎ ｄｅｐｅｎｄｅｎｔ ｋｉｎａｓｅ1

The present invention is a method of controlling the protein expression by making a DNA fragment complementary to the mRNA produced when the gene expression of cyclin dependent kinase1 acting on cell cycle control cellular response.

The movement of MPF into the nucleus during prophase in vertebrates is essential for the induction and coordination of M phase (divided) events. Phosphorylation of cyclin B is an important part of nuclear translocation. The cyclin dependent kinase 1 acts only when the expression of cyclin B increases at the G2 / M phase. When their expression is inhibited, the cell cycle stops. In many cancer cells, this regulatory mechanism is often broken, and there is a way to control this cyclin dependent kinase that can effectively inhibit cell division.

Lee, M. G., Norbury, C. J., Spurr, N. K., Nurse, P. Regulated expression and phosphorylation of a possible mammalian cell-cycle control protein. (Letter) Nature 333: 676-679, 1988. Moore, J. D., Kirk, J. A., Hunt, T. Unmasking the S-phase-promoting potential of cyclin B1. Science 300: 987-990, 2003.

Synthesis and Dosing of Antisense DNA to mRNA to Reduce the Expression of Cyclin-dependent Kinase1

Eighteen DNA nucleotides are synthesized for the front, middle and back of the gene sequence for mRNA. Dilute it and put it into cells or tissues.

1. The knock down effect can be augmented by looking at interference using DNA that is more stable than the conventional micro RNA method.

2. By controlling the expression level of cyclin dependent kinase1, it can be the basis to control various cancers and control cell proliferation and division.

Fig. 1 Complementary 18 base pairs for mRNA coding region of cyclin dependent kinase1
[Figure 2] KEGG pathway photo of cyclin dependent kinase1

The reason for using 18 base pairs in FIG. 1 is that there are 3 billion base pairs in the human gene. In order to induce specific binding in these base pairs, there must be at least 14 specific lengths of nucleotides, i.e., one in the number of fourteenth-squared cases. Therefore, 18base pair was used more safely. By targeting these DNA oligomers at the front, middle, and end of the mRNA, it is possible to appropriately control the expression level by selecting the one with the highest knockdown efficiency or the lowest efficiency. At this time, since 18base pair is a short strand, it can be ordered like PCR primer synthesis.

Claims (4)

A method for reducing the expression level of the protein in cultured cells, tissues, and in vivo by making a DNA oligomer of 10 base pairs or more complementary to the mRNA of the cyclin dependent kinase1 protein or its application The method of claim 1 wherein the DNA oligomer sequence of 18 base pair
3'-caaactttgacgagcgtg-5 '(complementary DNA strand to front)
3'-catttaatattttaacat-5 '(complementary DNA strand to middle)
3'-cagaaggtgtgtatgaaa-5 '(complementary DNA strand to the back) The method of preparing a DNA oligomer complementary to cyclin dependent kinase1 mRNA is based on the use of PCR primers. The experimental device or method according to claim 1, wherein the expression level of the cyclin dependent kinase protein is regulated through a DNA oligomer complementary to the cyclin dependent kinase1 mRNA to regulate the signaling system and cell cycle associated with the protein.
KR1020110136975A 2011-12-19 2011-12-19 Dna oligomer that binds as an antisense to the cyclin dependent kinase1 mrna KR20130069924A (en)

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KR1020110136975A KR20130069924A (en) 2011-12-19 2011-12-19 Dna oligomer that binds as an antisense to the cyclin dependent kinase1 mrna

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KR1020110136975A KR20130069924A (en) 2011-12-19 2011-12-19 Dna oligomer that binds as an antisense to the cyclin dependent kinase1 mrna

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KR20130069924A true KR20130069924A (en) 2013-06-27

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200062083A (en) 2018-11-26 2020-06-03 국립암센터 A method for screening a therapeutic agent for cancer using binding inhibitor of Cyclin-dependent kinase 1(CDK1) - Cyclin B1
KR20200062460A (en) 2018-11-26 2020-06-04 국립암센터 A method for screening a therapeutic agent for cancer using binding inhibitor of Cyclin-dependent kinase 1(CDK1) - Cyclin B1

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200062083A (en) 2018-11-26 2020-06-03 국립암센터 A method for screening a therapeutic agent for cancer using binding inhibitor of Cyclin-dependent kinase 1(CDK1) - Cyclin B1
KR20200062460A (en) 2018-11-26 2020-06-04 국립암센터 A method for screening a therapeutic agent for cancer using binding inhibitor of Cyclin-dependent kinase 1(CDK1) - Cyclin B1

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