• BMB Reports
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• v.41 no.2
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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.

• Bulletin of the Korean Chemical Society
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• v.33 no.1
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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.

• Bulletin of the Korean Chemical Society
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• v.33 no.11
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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.

• Bulletin of the Korean Chemical Society
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• v.35 no.9
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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.

• Journal of the Korean Electrochemical Society
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• v.19 no.4
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• pp.134-140
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• 2016

An aptamer-based biosensor using a new redox indicator has been examined for the electrochemical detection of thrombin. The aptamer modified primary aliphatic amine was covalently immobilized onto poly-(5,2':5',2"-terthiophene-3'-carboxylic acid) (polyTTCA) layer. Tetrabromophenolphthalein ethyl ester (KTBPE) was interacted to aptamer and used as an electrochemical indicator. Prior to the detection, the oxidation reaction of KTBPE onto aptamer modified layer was also investigated using differential pulse voltammetry. The characterization of the final sensor (KTBPE/aptamer -polyTTCA) was performed by voltammetry, QCM, and ESCA. After binding of thrombin onto KTBPE/aptamer based sensor, the peak signal of KTBPE was gradually decreased. The sensor exhibited a dynamic range between 10.0 and 100.0 nM with the detection limit of $1.0{\pm}0.2nM$.

• Bulletin of the Korean Chemical Society
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• v.34 no.3
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• pp.721-722
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• 2013

• Bulletin of the Korean Chemical Society
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• v.35 no.6
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• pp.1867-1870
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• 2014

• KSBB Journal
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• v.26 no.4
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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.

• Journal of Life Science
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• v.12 no.1
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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.