TY - JOUR
T1 - A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery
AU - Park, Sang Ryul
AU - Hauver, Jesse
AU - Zhang, Yunxiang
AU - Revyakin, Andrey
AU - Coleman, Robert A.
AU - Tjian, Robert
AU - Chu, Steven
AU - Pertsinidis, Alexandros
N1 - Funding Information:
A.P. and S.R.P. thank Carla Inoue for assistance with preparation of transcription factors, M. Vrljic and S. Sivasankar for multiple useful discussions, and Wen-Tau Juan for sharing unpublished results on grafting DNA polymer brushes. We also thank Gerry Hammer for XPS analysis of the PEG-passivated surfaces. The surface analysis experiments done at NESAC/BIO were supported by NIBIB grant EB-002027. This work was funded by the National Institutes of Health with a grant to R.T. and S.C. ( 5P01CA112181-05 ), by DARPA ( W911NF-06-1-0122 ), NSF ( 0647161 ) and NASA ( NNX07AK54G ) with grants to S.C., and the Louis V. Gerstner, Jr. Young Investigators Fund (A.P.), a National Cancer Institute grant ( P30 CA008748 ) and a National Institutes of Health (NIH) Director's New Innovator Award (1DP2GM105443-01; A.P.).
Publisher Copyright:
© 2020 Elsevier Ltd
PY - 2020/12/1
Y1 - 2020/12/1
N2 - Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.
AB - Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.
KW - PEG
KW - Pre-Initiation Complex
KW - RNA Polymerase
KW - general transcription factors
KW - pol II
KW - polyethylene glycol
KW - polymer brushes
KW - single-molecule assays
KW - surface passivation
KW - transcription
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U2 - 10.1016/j.str.2020.07.009
DO - 10.1016/j.str.2020.07.009
M3 - Article
C2 - 32763141
AN - SCOPUS:85089978166
SN - 0969-2126
VL - 28
SP - 1337-1343.e4
JO - Structure
JF - Structure
IS - 12
ER -