• Bulletin of the Korean Chemical Society
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• 제32권1호
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• pp.247-250
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• 2011

We designed an effective screening method for double strand DNA (dsDNA) binders using DNA-modified magnetic particles. Hairpin DNA was immobilized on the surface of magnetic particle for a simple screening of dsDNA binding materials in a solution containing various compounds. Through several magnetic separation and incubation processes, four DNA-binding materials, DAPI, 9AA, AQ2A, and DNR, were successfully screened from among five candidates. Efficiency of screening was demonstrated by HPLC analysis using a C2/18 reverse-phase column. In addition, their relative binding strengths were verified by measuring the melting temperature ($T_m$). If hairpin DNA sequence is modified for other uses, this magnetic bead-based approach can be applied as a high-throughput screening method for various functional materials such as anti-cancer drugs.

• 한국양식학회지
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• 제9권1호
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• pp.93-100
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• 1996

물고기에서 효과적인 발현 vector의 구성을 위해, 미꾸라지 MAR로부터 ARS를 cloning하여, 그 염기서열을 분석하였다. 총 443 염기들로 구성된 미꾸라지의 ARS는 다른 여러종들의 DNA 복제원점에서 나타나는 것 처럼, AT가 풍부하고, ARS consensus sequences, topoi-somerase II consensus sequences, 그리고 A 흑은 T-box등을 포함하고 있다. 그리고 그 DNA 단편은 복제원점에서 일반적인 양상으로 나타나는 반복적인 inverted sequence들을 가지고 있고, 5개의 가능한 hairpin loop 구조들을 내포하고 있다. 이들 구조는 DNA 복제개시에 관여하는 단백질들의 인지부위로 작용할 것으로 생각된다.

• BMB Reports
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• 제41권11호
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• pp.778-783
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• 2008

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in its 5' untranslated region (5'-UTR). To investigate the role of the hairpins in replication of TYMV, mutants lacking one or both of the two hairpins were constructed. The TYMV constructs were introduced into Chinese cabbage by an Agrobacterium-mediated T-DNA transfer method, called agroinfiltration. Analysis of total RNA from agroinfiltrated leaves showed that replication of the mutant TYMV RNA lacking both hairpins was about 1/100 of wild type. This mutant was also impaired in systemic spread. Deletion analysis of each hairpin revealed that both hairpins were needed for maximal replication. The deletion analysis along with sequence modification of the hairpin structure indicates that the second hairpin plays a role in efficient long-distance systemic movement of TYMV.

• BMB Reports
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• 제36권2호
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• pp.201-206
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• 2003

Two lymphoid-specific proteins, RAG1 and RAG2, are required for the initiation of the V(D)J recombination in vitro. The V(D)J cleavage that is mediated by RAG proteins at the border between the coding and signal sequences results in the production of a hairpin at the coding end and a double-stranded break at the signal end. Two hairpin coding ends are re-opened, modified, and sealed; whereas, the signal ends are directly ligated. Here I report that only RAG1 can carry out a distinct endonucleolytic activity in vitro using an oligonucleotide substrate that is tethered by a short single-stranded DNA. The purified RAG1 protein alone formed a nick at the near position to the recombination signal sequence. This endonucleolytic activity was eliminated by immunoprecipitation using the RAG1-specific antibody, and required the 3'-hydroxy group. All of the RAG1 mutants that were incapable of the nick and hairpin formation in the V(D)J cleavage analysis also showed this new endonucleolytic activity. This suggests that the nicking activity that was observed might be functionally different from the nick formation in the V(D)J cleavage.

• 한국동물학회지
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• 제27권2호
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• pp.103-116
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• 1984

유전자 구조 및 유전정보 흐름의 차이로 인하여 고등생물의 유전자를 미생물에 직접 cloning하면 원하는 유전자 산물을 얻지 못하는 경우가 많다. 이것을 극복하기 위해서는 화학적인 방법으로 유전자를 합성하든지, 또는 역제효소를 사용하여 고등생물의 mRNA로부터 유전자를 합성하여 cloning하는 방법을 사용한다. 본 연구에서는 oligo(dT)-cellulose column 방법으로 순수분리한 plasmid pBR322의 Pst I site에 cloning하였다. 우선 AMV reverse transcriptase로 primary cDNA를 합성하고, 알칼리를 처리하여 주형 RNA를 제거했다. 이번에는 이 primary cDNA를 주형으로 Klenow enzyme과 reverse transcriptase를 차례로 처리하여 double stranded DNA를 합성하고, 이 때 5' end 근처에 형성되는 hairpin loop을 Sl nuclease로 제거했다. Terminal deoxynucleotidyl transferase를 사용하여, 합성된 dsDNA에는 poly(dC) track을, Pst I endonuclease를 처리한 plasmid DNA에서는 poly(dG) track을 각각 붙인다음 이들을 서로 annealing시키고 E. coli에 transformation시켜서 크기가 큰 plasmid를 갖는 clone을 cracking 방법으로 일처 선별하였다. 이렇게 선별된 clone을 in 냐셔 hybridization 방법으로 조사하여 globin DNA가 들어간 colony를 이차 선별하고 여러 restriction enzyme으로 잘라보아 globin DNA가 cloning된 것을 확인하였다. 토끼 hemoglobin으로 immunize한 rat (Wistar)에서 뽑은 제일차 혈청과 염소에서 뽑은 제이차 혈청의 antibody를 사용한 radioimmunoassay방법으로, cloning된 globin gene이 대장균내에서 발현되는 지의 여부를 살펴 보았는데, 박테리아의 $\\beta$-lactamase와 토끼의 globin이 결합된 chimeric protein이 대장균 내에서 다량 합성되며, 이 단백질은 토끼 hemoglobin의 antigenic determinant를 가지고 있음을 알 수 있었다.

• BMB Reports
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• 제38권6호
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• pp.676-682
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• 2005

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of $NaBH_4$, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.72-76
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• 2017

Detection of exon 19 deletion mutation in the epidermal growth factor receptor (EGFR) gene, which results in increased and sustained phosphorylation of EGFR, is important for diagnosis and treatment guidelines in non-small-cell lung cancer. Here, we have developed a simple and convenient detection system using the interaction between G-quadruplex and fluorophore thioflavin T (ThT) for discriminating EGFR exon 19 deletion mutant DNA from wild type without a label and quencher. In the presence of exon 19 deletion mutant DNA, the probe DNAs annealed to the target sequences were transformed into G-quadruplex structure. Subsequent intercalation of ThT into the G-quadruplex resulted in a light-up fluorescence signal, which reflects the amount of mutant DNA. Due to stark differences in fluorescence intensity between mutant and wild-type DNA, we suggest that the induced G-quadruplex structure in the probe DNA can report the presence of cancer-causing deletion mutant DNAs with high sensitivity.

• Bulletin of the Korean Chemical Society
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• 제35권12호
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• pp.3655-3657
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• 2014

• Bulletin of the Korean Chemical Society
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• 제33권12호
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• pp.4219-4222
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• 2012

• BMB Reports
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• 제41권4호
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• pp.294-299
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• 2008

Elevated expression of SMYD3 is a frequent genetic abnormality in several malignancies. Few studies knocking down SMYD3 expression in cervical carcinoma cells have been performed to date. In this paper, we established an inducible short hairpin RNA expression system to examine its role in maintaining the malignant phenotype of HeLa cells. After being induced by doxycycline, SMYD3 mRNA and protein expression were both reduced, and significant reductions in cell proliferation, colony formation and migration/invasion activity were observed in the SMYD3-silenced HeLa cells. The percentage of cells in sub-G1 was elevated and DNA ladder formation could be detected, indicating potent induction of apoptosis by SMYD3 knockdown. These findings imply that SMYD3 plays crucial roles in HeLa cell proliferation and migration/invasion, and that it may be a useful therapeutic target in human cervical carcinomas.