• 제목/요약/키워드: Thrombin aptamer

검색결과 9건 처리시간 0.033초

Detection for folding of the thrombin binding aptamer using label-free electrochemical methods

  • Cho, Min-Seon;Kim, Yeon-Wha;Han, Se-Young;Min, Kyung-In;Rahman, Md. Aminur;Shim, Yoon-Bo;Ban, Chang-Ill
    • BMB Reports
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    • 제41권2호
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    • pp.126-131
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    • 2008
  • The folding of aptamer immobilized on an Au electrode was successfully detected using label-free electrochemical methods. A thrombin binding DNA aptamer was used as a model system in the presence of various monovalent cations. Impedance spectra showed that the extent to which monovalent cations assist in folding of aptamer is ordered as $K^+$ > $NH_4^+$ > $Na^+$ > $Cs^+$. Our XPS analysis also showed that $K^+$ and $NH_4^+$ caused a conformational change of the aptamer in which it forms a stable complex with these monovalent ions. Impedance results for the interaction between aptamer and thrombin indicated that thrombin interacts more with folded aptamer than with unfolded aptamer. The EQCM technique provided a quantitative analysis of these results. In particular, the present impedance results showed that thrombin participates a folding of aptamer to some extent, and XPS analysis confirmed that thrombin stabilizes and induces the folding of aptamer.

Spectroscopic and Electrochemical Detection of Thrombin/5'-SH or 3'-SH Aptamer Immobilized on (porous) Gold Substrates

  • Park, Buem-Jin;Sa, Young-Seung;Kim, Yong-Hwan;Kim, Young-Hun
    • Bulletin of the Korean Chemical Society
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    • 제33권1호
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    • pp.100-104
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    • 2012
  • Thrombin is a serine protease that catalyzes the conversion of soluble fibrinogen to insoluble fibrin, and thus induces physiological and pathological blood coagulation. Therefore, it is important to detect thrombin in blood serum for purposes of diagnosis. To achieve this goal, it has been suggested that a 15-mer aptamer strongly binds with thrombin to form a G-quartet structure of the aptamer. Generally, 5'-end thiol-functionalized aptamer has been used as an anti-thrombin binder. Herein, we evaluate the possibility of utilizing a 3'-SH aptasensor for thrombin detection using SPR spectroscopy, and compare the enhancement of the electrochemical signal of the thrombin-aptamer bound on a porous gold substrate. Although the two aptamers have similar configurations, in SPR analysis, the 3'-SH aptamer was a effective aptasensor as well as 5'-SH aptamer. Results from electrochemical analysis showed that the porous gold substrate acted as a good substrate for an aptasensor and demonstrated 5-fold enhancement of current change, as compared to gold thin film.

Potential of Mean Force Simulation by Pulling a DNA Aptamer in Complex with Thrombin

  • Yang, Changwon;Kim, Eunae;Pak, Youngshang
    • Bulletin of the Korean Chemical Society
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    • 제33권11호
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    • pp.3597-3600
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    • 2012
  • Thrombin binding aptamter (TBA-15) is a 15-mer guanine-rich oligonucleotide. This DNA apamer specifically binds to the thrombin protein involved in blood coagulation. Using extensive umbrella sampling molecular dynamics simulation method at all atom level, we investigated the potential of mean force (PMF) upon pulling the DNA aptamer from the binding mode of aptamer/thrombin complex. From this calculation, the free energy cost for a full dissociation of this aptamer/protein complex is 17 kcal/mol, indicating a substantial binding affinity of TBA-15. Interestingly, this PMF reveals noticeable plateau regions along the pulling coordinate. Possible structural changes of this complex in the plateau were investigated in details.

Aptamer-Based Precipitation as an Alternative to the Conventional Immunoprecipitation for Purification of Target Proteins

  • Song, Seongeun;Cho, Yea Seul;Lee, Sung-Jae;Hah, Sang Soo
    • Bulletin of the Korean Chemical Society
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    • 제35권9호
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    • pp.2665-2668
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    • 2014
  • Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based precipitation assays (or simply aptamoprecipitation) for His-tagged proteins and thrombin to compare their purification efficiency with other conventional affinity precipitation methods. A crosslinking method was employed to immobilize thiol-functionalized aptamers onto the surface of polystyrene resins, enabling them to specifically bind to His-tag and to thrombin, respectively. The resulting aptamer-functionalized resins were successfully applied via a one-step experiment to purification of His-tagged proteins from complex E. coli and to thrombin extraction, exhibiting superior or at least comparable purification results to the conventional immobilized metal affinity precipitation or immunoprecipitation.

전기전도성 고분자 위에 고정된 압타머에 흡착된 테트라브롬페놀프탈레인 에틸 에스테르를 이용한 트롬빈 검출 (Thrombin Detection with Tetrabromophenolphthalein Ethyl Ester Adsorbed on Aptamer-attached Conductive Polymer)

  • 정새로미;노희복;심윤보
    • 전기화학회지
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    • 제19권4호
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    • pp.134-140
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    • 2016
  • 새로운 산화환원 표지자를 이용한 압타머 기반의 전기화학적 트롬빈 검출 바이오 센서를 개발하였다. 1차 지방족 아민(primary aliphatic amine) 으로 개질한 압타머를 전기 전도성 고분자 poly-(5,2':5',2"-terthiophene-3'-carboxylic acid) (polyTTCA) 층 위에 공유결합을 통해 고정하여 센서 표면을 개질하였다. Tetrabromophenolphthalein ethyl ester (KTBPE)를 압타머와 상호 작용시켜 전기화학적인 산화환원 표지자로 사용하였다. 압타머로 개질한 층 위에 KTBPE의 산화반응을 differential pulse voltammetry (DPV)를 사용하여 조사하였으며, 최종 센서의 특성은 voltammetry, QCM, and ESCA 를 사용하여 조사하였다. KTBEF와 압타머 센서와 반응 후, KTBPE의 산화 피크는 감소하였다. 센서의 선형 동적 범위는 10.0 ~ 100.0 nM 이었으며, 이 때 검출 한계는 $1.0{\pm}0.2nM$이었다.

앱타머와 단백질간 가교를 이용한 바이오마커 진단 방법 개발 (The Method Development for Biomarker Diagnosis Based on the Aptamer-protein Crosslink)

  • 이보람;김진우;김병기
    • KSBB Journal
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    • 제26권4호
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    • pp.352-356
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    • 2011
  • The detection of biomarkers is an important issue for disease diagnosis. However, many systems are not suitable to detect the biomarker itself directly. For direct detection of biomarker proteins in human serum, a new affinity-capture method using aptamers combined with the mass spectrometry was suggested. Since signals from protein samples cannot be amplified, modified chromatin immunoprecipitation (ChIP) and subsequent cross-linking with formaldehyde between aptamers and target proteins were used not to lose the captured target proteins, which allowed us to perform a harsh washing step to remove the non-specifically bound proteins. As a model system, a thrombin aptamer was used as a bait and thrombin as a target protein. Using our modified ChIP and affinity-capture method, non-specific binding proteins on the beads decreased significantly, suggesting that our new method is efficient and can be applied to developing diagnosis systems for various biomarkers.

Aptamers (nucleic acid ligands) for trypsin-like serine proteases

  • Gal, Sang-Wan;Jeong, Yong-Kee;Satoshi Nishikawa
    • Journal of Life Science
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    • 제12권1호
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    • pp.14-18
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    • 2002
  • Subpopulations of nucleotides that bind specifically to a variety of proteins have been isolated from a population of random sequence RNA/DNA molecules. Roughly one in $10^{13}$ random sequence RNA/DNA molecules folds in such a way as to create a specific binding site for small ligands. Since the development of in vitro selection procedure, more than 50 nucleic acid ligands (aptamers) have been isolated. These molecules are very useful for the study of molecular recognition between nucleic acid and protein/organic compound. In addition to these basic studies this method gives us a dream to produce new drugs against several diseases. We focused on several aptamers which specifically binds to trypsin-like serine proteases (thrombin, human neutrophil elastase, activated protein C and NS3 protease of human hepatitis C virus) and want to introduce their structural characteristics and some functions.

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