Fig. 1: Chemical labeling of dCas9-Halo for selective G4-targeting. | Nature Communications

Fig. 1: Chemical labeling of dCas9-Halo for selective G4-targeting.

From: Chemically modified CRISPR-Cas9 enables targeting of individual G-quadruplex and i-motif structures, revealing ligand-dependent transcriptional perturbation

Fig. 1: Chemical labeling of dCas9-Halo for selective G4-targeting.The alternative text for this image may have been generated using AI.

a Schematic representation of a G-Tetrad via Hoogsteen base pairing with central cation (K+) (modified from PDB file:6W9P) (left) and G-tetrad stacking to form G4 structure (modified from PDB file:6W9P using Protein Imager96) (right). b Schematic overview of ATENA: dCas9-Halo fusion protein functionalized with chloroalkane-modified G4 ligands (created with BioRender, https://BioRender.com/hgoel9a) enables single G4-targeting through sgRNA guidance. c Chemical structure of chloroalkane-modified PyPDS, where n indicates the different PEG linker lengths (Cl-PyPDSn). d Chemical structure of chloroalkane-modified PhenDC3 (Cl-PhenDC3n), where n indicates the different PEG linker lengths (Cl-PhenDC3n). e Illustration (created with BioRender, https://BioRender.com/vmzonqy) of the competition assay workflow to test the binding ability of Cl-PyPDSn probes to dCas9-Halo purified recombinant protein. f SDS-PAGE gel of the Cl-PyPDSn competition assay shows each sample’s fluorescent level acquired in the TAMRA channel (542 nm) and the corresponding protein level (Coomassie staining): (n = 2). g Schematic representation (created with BioRender, https://BioRender.com/hirbnfn) of the dually labeled FRET oligos containing c-KIT2-G4-forming sequence bound by dCas9-Halo labeled with Cl-PyPDSn probes to study G4 stabilization with respect to the PEG linker length and sgRNA positioning. h ∆FRET efficiency of the decorated dCas9-PDS (with Cl-PyPDS2) complex targeting c-KIT2-G4. The values indicated were extrapolated from the band intensity measured in the Cy3 and Cy5 channels (Typhoon FLA 9500). The signals in both channels were normalized for the background and the sgRNA NTC control. These normalized fluorescence values were then used to calculate the ΔFRET efficiency for each sgRNA: FRET-Efficiency (E)+ligand sgRNAx – FRET-Efficiency (E)-ligand sgRNAx (n = 2). Data presented are the mean of n = number of independent experiments. Statistical significance was calculated using a two-tailed t test in GraphPad Prism; p-value: ns > 0.05, *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001. Source data are provided as a Source Data file.

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