Single-molecule dynamics in solution with anti-Brownian trapping

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have made progress on the following two areas during the past year a) Joint change-point detection of multiple measurement channels. During my previous position as an independent fellow at Princeton University, my lab have developed a new experimental platform called ABEL-FRET (Wilson and Wang, Nat. Methods 18, 816), which allows FRET dynamics of individual molecules to be monitored without tethering in solution. There are two major advantages of ABEL-FRET compared to conventional modalities: ultrahigh resolution of structural heterogeneities and simultaneous measurement of hydrodynamic parameters. Meanwhile, ABEL-FRET generates single-molecule time traces that are multidimensional in nature and poses new challenges for data analysis. In particular, a task of interest is to identify the locations of abrupt change points in the presence of measurement noise. These change points might occur in one measurement channel or in many channels in a correlated way. We developed a new analysis pipeline called MULLR (which stands for MUlti-channel Log-Likelihood Ratio test) to perform joint change point identification across multiple measurement channels. Being one of the first algorithms applicable to multichannel single-molecule time trace, MULLR is specifically designed to be model-free and can handle channels with different measurement statistics. We demonstrated MULLR on both simulated and experimental data and compared MULLR to alternative data processing strategies. This work further enhances ABEL-FRET to sense biomolecular processes at the single-molecule level. b) Single-molecule dissection of gRNA conformation during Cas9 holoenzyme assembly. Biomolecules carry out their function by cycling through a series of functional states. To better understand the structural-functional relations, it is of tremendous interest to probe structure at sequential stages of the functional states. We recently used the ABEL-FRET platform to probe the 3-end structure of CRISPR RNA (crRNA) at the single-molecule level as it assembles into the Cas9 holoenzyme. For every molecule, its assembly state is unambiguously determined using hydrodynamic profiling and its 3-end structure is probed by a pair of strategically placed FRET dyes. Strikingly, we discovered structural heterogeneity and dynamics at every stage of the assembly pathway that is, crRNA, guide RNA (gRNA, or crRNA-tracrRNA hybrid), Cas9-gRNA complex and Cas9-gRNA bound with substrate DNA, highlighting the importance of RNA structural diversity. Current work focuses on using RNA structural prediction tools to generate structural models consistent with single-molecule FRET measurements and devising plausible pathways of Cas9 holoenzyme assembly. This work could potentially shed light on fundamental biophysical principles of Cas9-gRNA recognition