The single-stranded DNA aptamer-binding site of human thrombin.

Paborsky, Lisa R.; McCurdy, Sarah N.; Griffin, Linda C.; Toole, John J.; Leung, Lawrence L.K.

doi:10.1016/s0021-9258(19)36856-5

“…However, in the presence of thrombin the aptamer forms a G-quartet binding to the thrombin [52][53][54][55]. The 15-mer single-stranded DNA aptamer (5 -GGTTGGTGTGGTTGG-3 ) binds to the fibrinogen recognition sites with a K d of 26 nM [56,57]. Previous research has studied the influence of the aptamer immobilization to solid surfaces and optimized binding assay conditions [58,59].…”

Section: Introductionmentioning

confidence: 99%

Aptamer Functionalized Lipid Multilayer Gratings for Label-Free Analyte Detection

Prommapan

¹

,

Brljak

²

,

Lowry

³

et al. 2020

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Lipid multilayer gratings are promising optical biosensor elements that are capable of transducing analyte binding events into changes in an optical signal. Unlike solid state transducers, reagents related to molecular recognition and signal amplification can be incorporated into the lipid grating ink volume prior to fabrication. Here we describe a strategy for functionalizing lipid multilayer gratings with a DNA aptamer for the protein thrombin that allows label-free analyte detection. A double cholesterol-tagged, double-stranded DNA linker was used to attach the aptamer to the lipid gratings. This approach was found to be sufficient for binding fluorescently labeled thrombin to lipid multilayers with micrometer-scale thickness. In order to achieve label-free detection with the sub-100 nm-thick lipid multilayer grating lines, the binding affinity was improved by varying the lipid composition. A colorimetric image analysis of the light diffracted from the gratings using a color camera was then used to identify the grating nanostructures that lead to an optimal signal. Lipid composition and multilayer thickness were found to be critical parameters for the signal transduction from the aptamer functionalized lipid multilayer gratings.

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“…Both studies suggested that aptamers form a target binding complex in coordination with divalent ions. Many aptamers such as thrombin-binding (Paborsky et al 1993), hematoporphyrin (Okazawa et al 2000), HIV-1 integrase (de Soultrait et al…”

Section: Designing An Aptamer Librarymentioning

confidence: 99%

Selection and characterisation of single-stranded DNA aptamers for triclosan

Li¹

0

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<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>

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“…Aptamers can be developed in vitro against a seemingly unlimited range of targets. To date, specific aptamers against highly diverse-range targets have been developed, such as ions (Ciesiolka et al 1995), small molecules (Li et al 1996), neurotransmitters (Mannironi et al 1997), organic pollutants (Joeng et al 2009;Jo et al 2011), drugs (Lato et al 1995;Wallis et al 1997), peptides (Welch et al 1997;Fukusaki et al 2001) , proteins (Paborsky et al 1993;Mendonsa and Bowser 2004), and even complex cells or tissues (Daniels et al 2003;Mallikaratchy et al 2007). Aptamers are stable under a wide range of temperature and buffer conditions and are resistant to harsh treatments such as physical or chemical denaturation without any significant loss of activity.…”

Section: Aptamers Versus Antibodiesmentioning

confidence: 99%

“…Recent studies have demonstrated that QDs can be used in the construction of aptasensors (Levy et al 2005;Choi et al 2006;Huang et al 2006;Liu et al 2011) This method makes use of the fact that many aptamer sequences (e.g. thrombin-binding, hematoporphyrin, HIV-1 integrase and ATP aptamers) form G-quadruplex structures upon binding to their targets (Paborsky et al 1993;Huizenga and Szostak 1995;Okazawa et al 2000;de Soultrait et al 2002). Thus, the incorporation of hemin molecules into such G-quadruplex structures can often lead to the formation of catalytically-active oligonucleotides, commonly known as DNAzymes, which are able to mimic horseradish peroxidase by catalysing the oxidation of luminol by H2O2, with the concomitant generation of chemiluminescence (Travascio et al 1998;Travascio et al 1999).…”

Section: Quantum Dot-based Fluorescent Aptasensorsmentioning

confidence: 99%

Selection and characterisation of single-stranded DNA aptamers for triclosan

Li¹

0

View full text Add to dashboard Cite

<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>

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The single-stranded DNA aptamer-binding site of human thrombin.

Cited by 185 publications

References 26 publications

Aptamer Functionalized Lipid Multilayer Gratings for Label-Free Analyte Detection

Aptamer Functionalized Lipid Multilayer Gratings for Label-Free Analyte Detection

Selection and characterisation of single-stranded DNA aptamers for triclosan

Selection and characterisation of single-stranded DNA aptamers for triclosan

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