High-throughput nitrogen-vacancy center imaging for nanodiamond photophysical characterization and pH nanosensing
Nanoscale Royal Society of Chemistry 12:42 (2020) 21821-21831
Abstract:
The fluorescent nitrogen-vacancy (NV) defect in diamond has remarkable photophysical properties, including high photostability which allows stable fluorescence emission for hours; as a result, there has been much interest in using nanodiamonds (NDs) for applications in quantum optics and biological imaging. Such applications have been limited by the heterogeneity of NDs and our limited understanding of NV photophysics in NDs, which is partially due to the lack of sensitive and high-throughput methods for photophysical analysis of NDs. Here, we report a systematic analysis of NDs using two-color wide-field epifluorescence imaging coupled to high-throughput single-particle detection of single NVs in NDs with sizes down to 5-10 nm. By using fluorescence intensity ratios, we observe directly the charge conversion of single NV center (NV- or NV0) and measure the lifetimes of different NV charge states in NDs. We also show that we can use changes in pH to control the main NV charge states in a direct and reversible fashion, a discovery that paves the way for performing pH nanosensing with a non-photobleachable probe.Closing and opening of the RNA polymerase trigger loop
Proceedings of the National Academy of Sciences National Academy of Sciences 117:27 (2020) 15642-15649
Abstract:
The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins
(2020)
Abstract:
ABSTRACT
Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli . The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction
Scientific Reports Nature Research 9 (2019) 16219
Confinement-free wide-field ratiometric tracking of single fluorescent molecules
Biophysical Journal Elsevier 117:11 (2019) 2141-2153