Professor Achillefs Kapanidis

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.

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.