Dataset Information

Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes.

ABSTRACT: RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2'-positions of participating guanosines with fluorophores. Importantly, this does not interfere with the required anti conformation of the nucleobase in a quadruplex with parallel topology. Sequential click reactions on the solid phase and in solution using a stop-and-go strategy circumvented the issue of unselective cross-labeling. We exemplified the method on a series of sequences under different assay conditions. In contrast to the commonly used end-labeling approach, our internal labeling strategy would also allow the study of G-quadruplex formation in long functional RNAs.

SUBMITTER: Hagen T

PROVIDER: S-EPMC6648711 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes.

Hagen Timo T Malinowska Anna L AL Lightfoot Helen L HL Bigatti Martina M Hall Jonathan J

ACS omega 20190514 5

RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2'-positions of participating guanosines with fluorophores. I ...[more]

PMID: 31459936

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Project description:The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Dataset Information

Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes.

Publications

Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes.

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