• 대한전기학회논문지:전기물성ㆍ응용부문C
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• 제53권5호
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• pp.286-292
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• 2004

In this study, an integrated microelectrode array was fabricated on glass slide using microfabrication technology. Probe DNAs consisting of mercaptohexyl moiety at their 5-end were spotted on the gold electrode using micropipette or DNA arrayer utilizing the affinity between gold and sulfur. Cyclic voltammetry in 5mM ferricyanide/ferrocyanide solution at 100 ㎷/s confirmed the immobilization of probe DNA on the gold electrodes. When several DNAs were detected electrochemically, there was a difference between target DNA and control DNA in the anodic peak current values. It was derived from specific binding of Hoechst 33258 to the double stranded DNA due to hybridization of target DNA. It suggested that this DNA chip could recognize the sequence specific genes. It suggested that multichannel electrochemical DNA microarray is useful to develop a portable device for clinical gene diagnostic System.

• 생명과학회지
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• 제15권6호
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• pp.935-940
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• 2005

Coumarine이 부착된 인산결합 단백질 (PBP-MDCC)의 형광변화가 뉴클레오사이드 삼인산 가수분해과정에서 배출된 무기 인산의 양을 측정하기 위해 관찰되었다. PBP-MDCC 정제후, 형광 방출 스펙트럼은 형광세기가 PBP-MDCC의 몰비을로 약 $70\%$까지 직선형태로 증가하는 것을 보였다. 형광 신호와 인산 기준물질과의 상호관계 측정이 인산 농도-형광세기 표준곡선을 구하기 위하여 stopped-flow 기구에서 행하여졌다. dTTP 가수분해로 부터 나오는 에너지를 이용하여 이중나선 DNA를 풀어주는 단백질인 T7박테리오파지 나선효소를 dPTT라 반응 시켰을 때, 형광변화를 배출된 인산의 양으로 전환할 수 있었다. 인산 배출 결과는 단일가닥 Ml3 DNA가 T7나선 효소에 의한 dTTP가수분해반응을 여러배 증가시키는 것을 보인다. 뉴클레오타이드 삼인산 가수분해 반응에 있어서 종말점 분석 대신에, PBP-MDCC에 의한 연속적인 인산 배출 분석이 배출된 인산을 측정하는데 있어서 쉽고 편리한 방법임을 보였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권4호
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• pp.263-267
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• 2003

Magnetic nanoparticles prepared from an alkaline solution of divalent and trivalent iron ions could covalently bind protein via the activation of Nethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC). Trypsin and avidin were taken as the model proteins for the formation of protein-nanoparticle conjugates. The immobilized yield of protein increased with molar ratio of EDC/nanoparticie. Higher concentrations of added protein could yield higher immobilized protein densities on the particles. In contrast to EDC, the yields of protein immobilization via the a ctivation of cyanamide were relatively lower. Nanoparticles bound with avidin could attach a single-stranded DNA through the avidin-biotin interaction and hybridize with a DNA probe. The DNA hybridization was confirmed by fluorescence microscopy observations. Immobilized DNA on nanoparticles by this technique may have widespread applicability to the detection of specific nucleic acid sequence and targeting of DNA to particular cells.

• 대한화학회지
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• 제46권2호
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• pp.151-156
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• 2002

Fucoidan은 갈조류에 다량 함유되어있는 다당류로서 항암작용, 항혈응고작용, 항혈전작용, 항염증작용, 항virus작용과 같은 생리활성작용력을 가지고 있어 최근 많은 연구가 수행되어지고 있다. 본 논문은 갈조류에서 fucoidan을 추출 하여 그 항암활성을 SV40 DNA replication assay, RPA-ssDNA binding assay, 그리고 MCF7 cell growth inhibition assay를 이용하여 알아보았으며 이 항암활성은 fucoidan의 황산기가 중요한 역할을 하고 있음을 알아내었다. 화학적으로 황산기의 함유량을 증가시킨 fucoidan이 황산기의 함유량을 제거시킨 fucoidan보다 항암활성이 뛰어났고, 이는 RPA의 ssDNA 결함력을 떨어 EM리기 때문이라 예측되어 진다. 본 연구는 한국산 갈조류에서 추출한 황산기를 함유한 fucoidan의 항암활성능력을 보여주었다.

• 한국미생물·생명공학회지
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• 제50권3호
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• pp.328-336
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• 2022

Aptamers are short, chemically synthesized, single-stranded DNA or RNA oligonucleotides that fold into unique three-dimensional structures. In this study, we aim to determine the antibiofilm activity and binding specificity of the six polyclonal DNA aptamers (S15K3, S15K4, S15K6, S15K13, S15K15, and S15K20) on Staphylococcus aureus BPA-12 and Escherichia coli EPEC 4. Aptamer S15K6 showed the highest percentage of antibiofilm activity against S. aureus BPA-12 (37.4%) as shown by the lowest OD570 value of 0.313. Aptamer S15K20 showed the highest percentage of antibiofilm activity against E. coli EPEC 4 (15.4%) as shown by the lowest OD570 value of 0.515. Aptamers S15K13 and S15K20 showed antibiofilm activities against both S. aureus BPA-12 and E. coli EPEC4, and thus potentially have broad reactivity. Furthermore, based on the binding capacity and Kd values from our previous study, the binding specificity assay of selected polyclonal DNA aptamers (S15K3 and S15K15) against S. aureus BPA-12, E. coli EPEC 4, S. aureus BPA-6, S. agalactiae, E. coli MHA-6, and Listeria monocytogenes were performed using qPCR. Aptamers S15K3 and S15K15 showed specific binding to S. aureus BPA-12, E. coli EPEC 4, S. aureus BPA-6, and S. agalactiae, but could not bind to E. coli MHA-6 and L. monocytogenes. Therefore, this study showed that the polyclonal DNA aptamers have antibiofilm activity and were able to bind to S. aureus BPA-12 and E. coli EPEC 4 bacteria.

• BMB Reports
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• 제34권5호
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• pp.428-433
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• 2001

A defect in uvsC of Aspergillus nidulans caused high methyl methansulfonate (MMS)-sensitivity, hyporecombination, and a lack of UV induced mutation. The uvsC gene of Aspergillus nidulans shares a sequence similarity with the RAD51 gene of Saccharomyces cerevisiae. In this study, in vitro and in vivo tests were conducted in order to determine whether or not the UVSC protein had functional similarities to RAD51, the recombination enzyme in yeast. The purified recombinant UVSC protein, following expression in Escherichia coli, showed binding activity to single-stranded DNA (ssDNA), when both ATP and magnesium are present. In addition, ATPase activity was also demonstrated and its activity was stimulated in the presence of ssDNA. The UVSC protein that was expressed under the ADH promoter in S. cerevisiae suppressed in part the sensitivity to MMS of the rad51 null mutant. Similarly, when the uvsC cDNA was expressed from the nmt promoter, the MMS sensitivity of the rhp51 null mutant of Schizosaccharomyces pombe was partially complemented. These results indicate that the A. nidulans UVSC protein is a functional homologue of the RAD51 protein.

• Animal Bioscience
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• 제34권10호
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• pp.1579-1589
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• 2021

Objective: This study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions. Methods: A combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5' labelled with biotin/6-FAM to determine the binding potential and binding pattern. Results: We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between -1.17 to -26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5'-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×10⁶ spermatozoa could trap almost 80% from the suspension. Conclusion: The binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

• Journal of Life Science
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• 제11권2호
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• pp.111-115
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• 2001

This study was designed to clone the SNF2/SW12 helicase-related genes from the fission yeast Schizosaccha-romyces pombe and thereafter to elucidate the common functions of the proteins in this family. The $hrp^{2+}$gene was cloned by polymerase chain reaction amplification using degenerative primers from conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. Like other SNF2/SW12 family proteins, the deduced amino acid sequence of Hrp2 contains DNA-dependent ATPase/7 helicase domains as well as the chromodomain and the DNA binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-dinding protein 1), suggesting that Hrp2 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to control the gene expression. To characterize the function of Hrp2, 4 Uracil-Hrp2 fusion protein, it was purified near homogeneity by affinity chromatography on $Ni^{2+}$-NTA agarose, DEAE-Sepharose ion exchange arid Sephacryl S-200 gel filtration chromatographies. The purified fusion protein exhibited DNA-dependent ATPase activity, which was stimulated by both double-stranded and single-stranded DNA. To determine the steady-state level of $hrp^{2+}$ transcripts during growth, cells were cultured in medium and collected at every 2hr to prepare total RNAs. The northern blot analysis showed that the level of $hrp^{2+}$ transcripts reached its maximum before the cells entered the exponential growth phase and then decreased gradually, This result implies that Hrp2 may be required at early stages of cell growth.h.

• Journal of Microbiology
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• 제35권4호
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• pp.327-333
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• 1997

Although extensively characterized in human cells, no heterogeneous nuclear ribonucleoprotein(hnRNP) has been found in the fission yeast Schizosaccharomyces pombe which is amenable to genetic studies and more similar to mammals than Saccharomyces cerevisiae is in terms of RNA processing. As a first step to characterize hnRNPs from S. pombe, attempt was made to find human hnRNP A1 homologs from S. pombe. The RNA molecule (A1 winner) containing the consensus high-affinity hnRNP A1 binding site (UAGGGA/U) was synthesized in vitro and used in an ultraviolet(UV) light-induced protein-RNA cross-linking assay. A number of S, pombe proteins bound to the A1 winner RNA. An approximately 50-kDa protein(p50) cross-linked more efficiently to the A1 winner RNA than other proteins. The p50 protein did not cross-link to a nonspecific RNA, but rather to the A1-5’ SS RNA in which the consensus 5’ splice junction sites of S. pombe introns were abolished. This suggests that the p50 protein, however, did not bind to the single-stranded DNA to shich the human hnRNP A1 could bind and be eluted with 0.5M NaCl. Further analysis should reveal more features of this RNA-binding protein.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.88-92
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• 2005

Aptamer is the single-stranded oligonucleotide which binds to various target molecules such as proteins, peptides, lipids and small organic molecules with high affinity and specificity. DNA aptamers specific for the $17{\beta}-estradiol$ were selected by SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random DNA library. These DNA aptamers have a high affinity to $17{\beta}-estradiol$ as an endocrine disrupting chemical. Nanowell and $200{\mu}m$ gold electrode were used as substrate for DNA aptamer immobilization and electrochemical analysis. Especially, nanowell gold electrode was fabricated by e-beam lithography. The size of single nanowell is 130nm and 40,000 nanowells were deposited on one gold electrode. The immobilization method was based on the interaction between the biotinylated aptamer and streptavidin deposited on gold electrode previously. Immobilization procedure was optimized by surface plasma resonance (SPR) and electrochemical analysis. After the immobilization of DNA aptamer on streptavidin modified gold electrode, $17{\beta}-estradiol$ solution was treated on aptamer immobilized gold electrode. The current of gold electrode was decreased by the binding of $17{\beta}-estradiol$ to DNA aptamer immobilized on gold electrode. However, in negative control experiments of 1-aminoanthraquinone and 2-methoxynaphthalene, the current was rarely decreased. And more sensitive data was obtained from nanowell gold electrode comparing with $200{\mu}m$ gold electrode.