Development of biosensing technology using specific interaction of biomolecules with metal ions = 생체 물질과 금속 이온의 상호작용을 이용한 바이오 센싱 기술 개발

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In recent years, an intense interest has grown in the interactions of biomolecules such as nucleic acids and proteins with metal ions. Examples of such novel interactions include the specific binding of aptamers with metal ions and selective incorporation of metal ions as cofactors to promote the catalytic activities of nucleic acid enzymes (deoxyribozymes or ribozymes). Certain metal ions like $Hg^{2+}$, $Ag^{+}$, $Cu^{2+}$, $Ni^{2+}$, and $Co^{2+}$ are shown to specifically bind to natural or artificially modified nucleosides to form metal ion-mediated base pairs. In addition, on the basis of well-established binding modes between nucleic acids and metal ions, inorganic nanomaterials such as silver nanoclusters and quantum dots have been synthesized by utilizing nucleic acids as bio-mineralization templates. Likewise, proteins have been utilized to produce inorganic nanomaterials with the different shapes, sizes, and crystal structures. A representative example includes the protein-directed synthesis of fluorescent gold nanoclusters, which are prepared by the reduction capability of bovine serum albumin. Based on the aforementioned unique interactions between biomolecules and metal ions, we have developed a novel biosensing technology for metal ions, biological thiols, and drug molecules such as theophylline and coralyne. In chapter 2, we have designed a new strategy in which a polymerase enzyme is controlled to accomplish an unnatural extension reaction even at the mismatched site of a primer with template DNA. The validity of this novel concept has been systematically demonstrated by using metal ions ($Hg^{2+}$ and $Ag^{+}$ ions) to intentionally trigger an unusual illusionary polymerase activity at respective T-T and C-C mismatched primers on the basis of their specific interactions with the respective mismatched base pairs (T-T and C-C). Being different from previous efforts in which interactions of nucleic acids with metal ions were studied, the current investigation has probed these interactions in combination with polymerase activity. A novel strategy to construct molecular scale logic gates has been also developed based on the unnatural polymerase activity induced by metal ions. The successful operation of the key logic gates has been demonstrated by rationally designing primers and selecting the type of DNA polymerase employed. The most notable feature of the logic gates devised herein is their simplicity and cost-effective design since the only requirement for the construction of logic gates is the incorporation of a single mismatched base (T and C) at the 3’ end of the primer and the application of metal ions ($Hg^{2+}$ and $Ag^{+}$ ions). In chapter 3, a new technology that enables the reliable detection of silver ions has been developed. The method takes advantage of the unique fluorescence property of a mismatched pyrrolo-dC (PdC)-modified duplex DNA, which serves as the key detection component, and the specific interaction of this duplex with silver ions. The new sensing strategy exhibits high selectivity and sensitivity and it does not require the use of procedures to pre-incorporate fluorophore or quencher labels. The latter feature is one of the greatest merits of the new fluorescence-based method. In addition, the novel concept has been successfully extended to the detection of biological thiols that is based on their specific and tight binding to silver ions. To the best of our knowledge, this is the first study showing that a specific interaction of a metal ion (e.g., $Ag^{+}$ ions) with a fluorescent mismatched nucleobase pair can be employed as the basis for a new type of metal ion sensing system. As a result, the observations made in this study serve as the basis of new sensing strategies that utilize the optical and binding properties of nucleobases and/or their analogs. In chapter 4, a novel, label-free, fluorescent, turn-on sensor for biological thiol detection that uses highly fluorescent gold nanoclusters (AuNCs), prepared by a bovine serum albumin (BSA)-templated green synthetic route, has been developed. This assay relies on blocking $Hg^{2+}$ -induced quenching of fluorescent AuNCs, caused by metallophilic $Hg^{2+}-Au^+$ interactions, through selective coordination of biological thiols with $Hg^{2+}$ ions. Biogical thiols entrap added $Hg^{2+}$ ions via a robust Hg-S interaction. This phenomenon prevents $Hg^{2+}$ -induced quenching and results in high fluorescence from the AuNCs. By employing this turn-on sensor, biological thiols, such as cysteine (Cys), glutathione (GSH) and homocysteine (Hcy), have been successfully detected at concentrations as low as 8.3 nM for Cys, 9.4 nM for GSH, and 14.9 nM for Hcy. The diagnostic capability of this method has also been demonstrated by detecting biological thiols in human blood serum, showing the great potential in the practical applications. The observations made in this study should aid the design of other fluorescence-based turn-on biosensors that broaden the applicability of AuNCs. In chapter 5, we have devised a novel, label-free, fluorescent sensor for sensitive and selective detection of theophylline utilizing abasic-site-containing duplex DNA as dual purposes; one is the synthesis template for fluorescent silver nanoclusters and the other is the binding pocket for theophylline. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic-site-containing duplex DNA. In the absence of theophylline, silver ions can interact with the cytosine opposite the abasic site in duplex DNA, which allows the efficient formation of fluorescent silver nanoclusters. In contrast, the presence of theophylline, which pseudo base pairs with the cytosine opposite the abasic site and is stabilized by nucleobases flanking the abasic site in duplex DNA, inhibits silver ions from binding the cytosine nucleobase. Consequently, fluorescent silver nanoclusters cannot be formed. As a result, assay samples that do not contain target theophylline display an intense red fluorescence signal while those containing theophylline show a significantly reduced fluorescence signal. This difference can be easily detected even with the naked eye under a hand-held UV lamp. By employing this new fluorescent sensor, theophylline is successfully analyzed at the concentration as low as 1.8 μM with the high selectivity over the structurally related methylxanthine derivatives and other molecules present in serum. The diagnostic capability of this method is also demonstrated by detecting theophylline in human blood serum, showing its great potential in the practical applications. In chapter 6, a novel, label-free, fluorescent turn-on detection system for screening of homo-adenine binding molecules, which employs DNA-templated silver nanoclusters (DNA-AgNCs) as a key detection component, has been developed. The new strategy relies on the formation of Non-Watson-Crick homo-adenine DNA duplex through the high affinity interaction between adenine-rich DNA sequence and its binding molecule, which brings guanine-rich sequence in proximity to DNA-AgNCs. This phenomenon transforms the weakly fluorescent AgNCs into the highly emissive species, which results in the emission of bright red fluorescence. By using this turn-on assay, we have successfully identified a coralyne molecule, which is known to selectively bind to homo-adenine and subsequently trigger the formation of non-Watson-Crick homo-adenine DNA duplex. Importantly, this new method is well suited to high-throughput screening system for the identification of candidate molecules binding to homo-adenine because it is simply operated without the complicated modifications and technical expertise.
Park, Hyun Gyuresearcher박현규researcher
한국과학기술원 :생명화학공학과,
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학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2013.8 ,[xv, 144 p. :]


Biosensor; DNA; Metal ion; Nucleotide; Protein; 바이오센서; 디옥시리보핵산; 금속 이온; 뉴클레오타이드; 단백질

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