title: Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices.
abstract: Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.
authors.affiliation: Nano Science and Technology Program, Taiwan International Graduate Program, Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Department of Engineering and System Science, National Tsing Hua University, ESS New Building, 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan.
authors.forename: K K
authors.initials: KK
authors.lastname: Sriram
authors.affiliation: Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan.
authors.forename: Jia-Wei
authors.initials: JW
authors.lastname: Yeh
authors.affiliation: Nano Science and Technology Program, Taiwan International Graduate Program, Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Department of Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Road, Daan, Taipei 10617, Taiwan.
authors.forename: Yii-Lih
authors.initials: YL
authors.lastname: Lin
authors.affiliation: Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan.
authors.forename: Yi-Ren
authors.initials: YR
authors.lastname: Chang
authors.affiliation: Institute of Physics, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Research Centre for Applied Sciences, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Genomic Research Centre, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan cfchou@phys.sinica.edu.tw.
authors.forename: Chia-Fu
authors.initials: CF
authors.lastname: Chou
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors; D004247:DNA
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