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iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

ABSTRACT: Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.

SUBMITTER: Joshi BS

PROVIDER: S-EPMC11261617 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Joshi Bhagyashree S BS de Lannoy Carlos C Howarth Mark R MR Kim Sung Hyun SH Joo Chirlmin C

Nano letters 20240708 28

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual mo ...[more]

PMID: 38975639

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Project description:CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

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iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

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<i>iMAX</i> FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

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iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

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&lt;i&gt;iMAX&lt;/i&gt; FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

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OmicsDI is part of the ELIXIR infrastructure

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<i>iMAX</i> FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.