• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1149-1153
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• 2006

In this study, nuclease-resistant RNA aptamers that are specific for Jurkat T leukemia cells were selected by a subtractive systemic evolution of ligands by exponential enrichment (SELEX) method. A randomized nuclease-resistant RNA library was incubated with normal peripheral blood mononuclear cells (PBMC) in each round to preclude RNAs that recognize the common cellular components on the surface of normal and cancer cells. The precluded RNAs were used for the selection of Jurkat T cell-specific aptamers, and the specific RNAs were then gradually enriched from start to the following selections. After 16 rounds of the subtractive SELEX, the selected aptamers were found to preferentially bind to Jurkat T cells, but not to the normal PBMC, evidenced by fluorescence-activated cell sorting analysis. Thus, the subtractive SELEX can be used to identify ligands to cancer-specific biological markers without prior knowledge of the nature of markers. The aptamers could be applied to specific cell sorting, tumor therapy, and diagnosis, and moreover, to find cancer cell-specific markers.

• Molecules and Cells
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• 제37권10호
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• pp.742-746
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• 2014

The epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is specifically detected in most adenocarcinomas and cancer stem cells. In this study, we performed a Cell systematic evolution of ligands by exponential enrichment (SELEX) experiment to isolate the aptamers against EpCAM. After seven round of Cell SELEX, we identified several aptamer candidates. Among the selected aptamers, EP166 specifically binds to cells expressing EpCAM with an equilibrium dissociation constant (Kd) in a micromolar range. On the other hand, it did not bind to negative control cells. Moreover, EP166 binds to J1ES cells, a mouse embryonic stem cell line. Therefore, the isolated aptamers against EpCAM could be used as a stem cell marker or in other applications in both stem cell and cancer studies.

• Bulletin of the Korean Chemical Society
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• 제30권8호
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• pp.1827-1831
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• 2009

Ligand molecules that can recognize and interact with cancer cell surface marker proteins with high affinity and specificity should greatly aid the development of novel cancer diagnostics and therapeutics. HER-2/ErbB2/Neu (HER-2), a member of the epidermal growth factor receptor family, is specifically overexpressed on the surface of breast cancer cells and serves as both a useful biomarker and a therapeutic target for breast cancer. In this study, we aimed to isolate RNA aptamers that specifically bind to a HER-2-overexpressing human breast cancer cell line, SK-BR-3, using Cell-SELEX strategy. The selected aptamers showed strong affinity to SK-BR-3, but not to MDAMB- 231, a HER-2-underexpressing breast cancer cell line. In addition, we confirmed the specific targeting of HER-2 receptor by aptamers using an unrelated mouse cell line overexpressing human HER-2 receptor. The HER-2-targeting RNA aptamers could become a useful reagent for the development of breast cancer diagnostics and therapeutics.

• Molecules and Cells
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• 제39권11호
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• pp.807-813
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• 2016

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of $1.3{\times}10^6CFU/ml$. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate ($5{\times}10^4CFU/ml$). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of $2{\times}10^4CFU/mL$ of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

• Journal of Biosystems Engineering
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• 제39권2호
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• pp.111-114
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• 2014

Purpose: This review aims to introduce aptamers and the methods of its development to improve the sensitivity and selectivity to target bacteria. In this review, we have highlighted current developments and directions in the pathogen detection based on aptamers. Background: Aptamers, the specific nucleic acid sequences, can bind to targets with high affinity and specificity. Some of researches on the use of aptamers for the detection of pathogen have been reported in recent years. Aptamers have more applicability than antibodies for the development of pathogen detection using biosensor; such as easy to synthesis and labeling, lack of immunogenicity, and a low cost of production. However, only few reports on the development and use of aptamers for the detection of pathogen have been published. Review: Aptamers specific to pathogen are obtained by whole-cell systematic evolution of ligands by exponential enrichment (SELEX) process. SELEX process is composed of screening random oligonucleotide bound with target cells, multiple separation and amplification of nucleic acids, final identification of the best sequences. For improving those affinity and selectivity to target bacteria, optimization of multiple separating process to remove unbounded oligonucleotides from aptamer candidates and sorting process by flow cytometry are required.

• Genomics & Informatics
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• 제3권2호
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• pp.77-80
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• 2005

Targeting of complex system such as human cells rather than biochemically pure molecules will be a useful approach to massively identify ligands specific for the markers associated with human disease such as cancer and simultaneously discover the specific molecular markers. In this study, we developed in vitro selection method to identify nuclease-resistant nucleic acid ligands called RNA aptamers that are specific for human cancer cells. This method is based on the combination of the cell-based selection and subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. These aptamers will be useful for cancer-specific ligands for proteomic research to identify cancer-specific molecular markers as well as tumor diagnosis and therapy.

• Journal of Microbiology and Biotechnology
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• 제25권3호
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• pp.393-398
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• 2015

Aptamers are composed of single-stranded oilgonucleotides that can selectively bind desired molecules. It has been reported that RNA or DNA could act as not only a genetic messenger but also a catalyst in metabolic pathways. RNA aptamers (average sizes 40-50 bp) are smaller than antibodies and have strong binding capacities to target molecules, similar to antigenantibody interactions. Once an aptamer was selected, it can be readily produced in large quantities at low cost. The objectives of this study are to screen and develop aptamers specific to oral pathogens such as Porphyromonas gingivalis, Treponema denticola, and Streptococcus mutans. The bacterial cell pellet was fixed with formaldehyde as a target molecule for the screening of aptamers. The SELEX method was used for the screening of aptamers and a modified western blot analysis was used to verify their specificities. Through SELEX, 40 kinds of aptamers were selected and the specificity of the aptamers to the bacterial cells was confirmed by modified western blot analysis. Through the SELEX method, 40 aptamers that specifically bind to oral pathogens were screened and isolated. The aptamers showed possibility as effective candidates for the detection agents of oral infections.

• 한국콘텐츠학회논문지
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• 제21권4호
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• pp.825-832
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• 2021

본 연구는 치주질환의 주 원인균인 P. gingivalis에 선택적으로 작용하는 특이적 앱타머를 선별하고 선별된 앱타머와 결합하는 단백질 분자를 정제 및 동정함으로써 P. gingivalis에 관한 작용기전을 규명하고자 하였다. 이를 위하여 39개의 random sequence를 갖는 DNA library를 제조하여 SELEX 방법을 이용하여 P. gingivalis에 특이성을 가진 앱타머를 선별하였으며 PCR2.1 cloning vector를 활용한 cloning을 시행하여 염기서열을 분석했다. 8종의 각기 다른 염기서열을 가진 앱타머를 선별하였고 직접적으로 작용하는 단백질을 밝혀내고자 선별된 앱타머 중 APG-3를 이용하여 modified weston blot을 시행하고 단백질을 분석한 결과 P. gingivalis에 선택적으로 결합하는 11종의 단백질을 분리, 동정하였다. 이와 같은 결과로 앱타머가 치주질환의 원인균인 P. gingivalis의 당 대사 및 세포활성억제와 관련된 단백질에 선택적으로 결합하여 부착함으로써 치주질환의 진단을 위한 센서로 가능성을 제시했다.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1634-1639
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• 2006

Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

• KSBB Journal
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• 제20권2호
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• pp.88-92
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• 2005

인산화 과정은 serine, threonine, tyrosine에서 발생하는 생화학적 반응으로, 본 연구에서는 다양한 응용의 잠재능력이 있는 phosphoserine, phosphothreonine, phosphotyrosine에 대한 엡타머를 개발하였다. 우선 in vitro selection 방법에 의해 combinatorial chemistry로부터 얻어진 RNA library로부터 이들 phosphoamino acids와 친화도를 가지고 있는 엡타머를 찾아낼 수 있었다. 총 10번의 일련 과정을 통해 phosphoserine에 대해서 2.6 nM의 친화도를 가지고 있는 엡타머를 (SeA-06), phosphothreonine에 대해서는 2.7nM의 친화도를 가지고 있는 엡타머 (TrA-18)를 찾아낼 수 있었고, 이들의 RNA 2차 구조를 각각 예측하여 보았다. 그러나 phosphotyrosine의 경우 짧은 길이의 엡타머가 selection됨으로 내부적으로 구조를 가지는 엡타머는 얻을 수 없었다. o-phosphoserine에 대한 항체가 기존에 보고가 되었으나 유사한 구조를 지닌 o-phosphothreonine에도 비슷한 활성을 보여 이들을 구분할 수 있는 리간드를 찾기 힘들었으나, 본 연구에서는 엡타머를 사용한 특이성 조사에서도 서로를 극명하게 구별할 수 있음을 확인할 수 있었다. 이와 같이 발굴된 엡타머를 사용하여 biochip이나 Proteomics 분석 도구의 응용에 큰 기대효과를 제공할 수 있을 것이라 사료된다.