Real-Time Observation of G-Quadruplex Dynamics Using Single-Molecule FRET Microscopy

ABSTRACTThe CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

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Real-time observation of the dynamics of an individual rotaxane molecular shuttle using a single-molecule junction

Chem ◽

10.1016/j.chempr.2021.11.012 ◽

2021 ◽

Author(s):

Sujun Chen ◽

Dingkai Su ◽

Chuancheng Jia ◽

Yanwei Li ◽

Xingxing Li ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Molecule Junction ◽

Single Molecule Junction ◽

Time Observation ◽

Molecular Shuttle ◽

Real Time Observation

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Real Time Observation of Chemical Reactions of Individual Metal Nanoparticles with High-Throughput Single Molecule Spectral Microscopy

Analytical Chemistry ◽

10.1021/ac101933y ◽

2010 ◽

Vol 82 (20) ◽

pp. 8744-8749 ◽

Cited By ~ 32

Author(s):

Jing Cheng ◽

Yang Liu ◽

Xiaodong Cheng ◽

Yan He ◽

Edward S. Yeung

Keyword(s):

Metal Nanoparticles ◽

Real Time ◽

Chemical Reactions ◽

High Throughput ◽

Single Molecule ◽

Individual Metal ◽

Time Observation ◽

Real Time Observation

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Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

10.1101/048371 ◽

2016 ◽

Cited By ~ 1

Author(s):

Digvijay Singh ◽

Samuel H. Sternberg ◽

Jingyi Fei ◽

Jennifer A. Doudna ◽

Taekjip Ha

Keyword(s):

Real Time ◽

Single Molecule ◽

Dna Cleavage ◽

Genome Engineering ◽

Dissociation Rate ◽

Stable Complex ◽

Guide Rna ◽

Single Molecule Fret ◽

Protospacer Adjacent Motif ◽

Time Observation

Binding specificity of Cas9-guide RNA complexes to DNA is important for genome engineering applications, but how mismatches influence target recognition and rejection kinetics is not well understood. We used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence, but the dissociation rate greatly increases from < 0.006 s-1 to > 2 s-1 upon introduction of mismatches proximal to the protospacer adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 12 base pairs in length, which prevent DNA cleavage, still allow the formation of a stable complex (off-rate < 0.006 s-1), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome editing thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

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