title: Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries.
abstract: We employed modified glass nanocapillaries to investigate interactions between the RNA-binding protein, known as cell carcinoma antigen recognized by T cells-3 (SART3), and the noncoding spliceosome component, U6 small nuclear RNA (snRNA), at the single-molecule level. We functionalized the nanocapillaries with U6 snRNA fragments, which were hybridized to DNA molecules and then covalently attached to the nanocapillary surface. When transported through the modified nanocapillaries, two different SART3-derived constructs, HAT-RRM1-RRM2 and RRM1-RRM2, exhibited resistive ionic current pulses with different dwell times, which represented their different binding affinities to tethered U6 snRNAs. The dissociation constants (KD), estimated from the bias voltage dependence of translocation events, were approximately 1.9 μM and 201 μM for HAT-RRM1-RRM2 and RRM1-RRM2, respectively. These values were comparable to corresponding values obtained with isothermal titration calorimetry, demonstrating that the modified glass nanocapillaries are applicable to analyses of protein-ligand interactions at the single-molecule level.
authors.affiliation: Department of Physics, Yonsei University , Seoul, 03722, Republic of Korea.
authors.forename: Choongman
authors.initials: C
authors.lastname: Lee
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology , Seoul, 02792, Republic of Korea.
authors.forename: Joon Kyu
authors.initials: JK
authors.lastname: Park
authors.affiliation: Department of Physics, Yonsei University , Seoul, 03722, Republic of Korea.
authors.forename: Yeoan
authors.initials: Y
authors.lastname: Youn
authors.affiliation: Department of Physics, Yonsei University , Seoul, 03722, Republic of Korea.
authors.forename: Joo Hyoung
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Physics, Yonsei University , Seoul, 03722, Republic of Korea.
authors.forename: Kyo-Seok
authors.initials: KS
authors.lastname: Lee
authors.affiliation: Advanced Analysis Center, Korea Institute of Science and Technology , Seoul, 02792, Republic of Korea.
authors.forename: Nak-Kyoon
authors.initials: NK
authors.lastname: Kim
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology , Seoul, 02792, Republic of Korea.
authors.forename: Eunji
authors.initials: E
authors.lastname: Kim
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology , Seoul, 02792, Republic of Korea.
authors.forename: Eunice Eunkyeong
authors.initials: EE
authors.lastname: Kim
authors.affiliation: Department of Physics, Yonsei University , Seoul, 03722, Republic of Korea.
authors.forename: Kyung-Hwa
authors.identifier: 0000-0001-9186-3095
authors.initials: KH
authors.lastname: Yoo
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D004560:Electricity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D043942:Nanotubes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D010455:Peptides; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C416530:SART3 protein, human; C405486:U6 small nuclear RNA