Journal
ANALYTICAL CHEMISTRY
Volume 88, Issue 21, Pages 10675-10679Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.6b03127
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Funding
- National Natural Science Foundation of China (NSFC) [31470269]
- Youth Innovation Promotion Association CAS
- Chinese Academy of Sciences [KJZD-EW-TZ-L04]
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Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)(2)dppx](2+), by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)(2)dppx](2+) complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.
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