title: Streptavidin-triggered signal amplified fluorescence polarization for analysis of DNA-protein interactions.
abstract: Fluorescence polarization (FP) is a sensitive, robust, and homogeneous assay format, able to probe a diversity of biological molecules and their interactions. Herein, we describe a new FP strategy based on the use of streptavidin as a signal amplifier. Such signal amplified fluorescence polarization (SAFP) was used to monitor the binding affinity of human angiogenin and a single-stranded DNA aptamer. Streptavidin was bound to a biotinylated single-stranded DNA aptamer and the interaction between this complex and Alexa Fluor 488 labelled human angiogenin was measured. A dissociation constant of 135.3 ± 32.9 nM and a limit of detection of 6.3 nM were successfully extracted only when the FP signal was increased (without binding hindrance) via streptavidin. Moreover, the demonstrated approach was specific to target molecules without any non-specific binding. The streptavidin-triggered SAFP method unlike amplification strategies that utilize nanomaterials (such as graphene oxides, carbon nanotubes, and metal nanoparticles) is not compromised by fluorescence quenching, and it is able to operate within nanomolar concentration regimes. Furthermore, unlike the other FP signal amplification strategies that use dual binding DNA probes, the presented method is simple to implement with signal amplification only requiring the binding of streptavidin with biotinylated DNA. This method could be expanded to analyze molecular interactions and it may be a useful tool for FP measurement by reducing the concentration of rare and expensive protein samples.
authors.affiliation: Department of Biochemistry, Chungbuk National University, Cheongju 28644, Republic of Korea. hykim@chungbuk.ac.kr.
authors.forename: Jae-Won
authors.initials: JW
authors.lastname: Choi
authors.affiliation: Department of Biochemistry, Chungbuk National University, Cheongju 28644, Republic of Korea. hykim@chungbuk.ac.kr.
authors.forename: Byung-Gwan
authors.initials: BG
authors.lastname: Jo
authors.affiliation: Department of Chemistry & Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8039 Zürich, Switzerland. andrew.demello@chem.ethz.ch.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: deMello
authors.affiliation: Department of Bionano Technology, Hanyang University, Ansan 15588, Republic of Korea. jbchoo@hanyang.ac.kr.
authors.forename: Jaebum
authors.initials: J
authors.lastname: Choo
authors.affiliation: Department of Biochemistry, Chungbuk National University, Cheongju 28644, Republic of Korea. hykim@chungbuk.ac.kr.
authors.forename: Hak Yong
authors.initials: HY
authors.lastname: Kim
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis; D015374:Biosensing Techniques; D004247:DNA / Q000032:analysis*; D005454:Fluorescence Polarization; D006801:Humans; D011485:Protein Binding; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D004247:DNA; D019809:Streptavidin; C046366:angiogenin; D012259:Ribonuclease, Pancreatic