title: A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids.
abstract: A portable method of specific nucleic acid detection would be very useful for monitoring public health in a variety of settings for point-of-care and point-of-need testing. However, conventional methods for the detection of nucleic acids are not ideal for use in the field, as they require skilled operators and complex equipment. Here, we constructed a method for specific nucleic acid detection using a split G-quadruplex (Gq) structure that can recognize target nucleic acids without competitive reactions in a bimolecular reaction and directly produce a detectable signal based on peroxidase activity. We developed a single signal-transducing molecule with a split Gq-based DNA-nano tweezers (NT) structure that self-assembles from three single-stranded DNAs through simple mixing, and detects its target without requiring any washing steps. A model target, a partial norovirus mRNA (NV-RNA), was specifically recognized by the split Gq-based DNA-NT, causing it to undergo a structural change that restored its peroxidase activity. The peroxidase activity was measured by following the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the NV-RNA concentration. The lower detection limit was 4 nM. Our results demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT structure as a nucleic acid signal-transducing molecule in a homogenous assay format. Also the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device.
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nakatsuka
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan; Institute for Sustainable Sciences and Development, Hiroshima University, 1-3-2 Kagamiyama, Higashihiroshima, Hiroshima 739-8527, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Shigeto
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
authors.affiliation: Institute for Sustainable Sciences and Development, Hiroshima University, 1-3-2 Kagamiyama, Higashihiroshima, Hiroshima 739-8527, Japan. Electronic address: hisafuna@hiroshima-u.ac.jp.
authors.forename: Hisakage
authors.initials: H
authors.lastname: Funabashi
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D017250:Caliciviridae Infections / Q000821:virology; D003124:Colorimetry / Q000379:methods; D004277:DNA, Single-Stranded / Q000737:chemistry*; D054856:G-Quadruplexes; D006801:Humans; D057230:Limit of Detection; D029322:Norovirus / Q000302:isolation & purification*; D019095:Point-of-Care Systems; D012367:RNA, Viral / Q000032:analysis*
chemical_list: D004277:DNA, Single-Stranded; D012367:RNA, Viral
keywords: Homogeneous assay; Norovirus; Nucleic acid detection; Point-of-care testing; Signal-transducing molecule; Split G-quadruplex