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Capture of RNA G-quadruplex structures using an l-RNA aptamer.


ABSTRACT: G-quadruplexes (dG4 and rG4) are nucleic acid secondary structures formed by the self-assembly of certain G-rich sequences, and they have distinctive chemical properties and play crucial roles in fundamental biological processes. Small molecule G4 ligands were shown to be crucial in characterizing G4s and understanding their functions. Nevertheless, concerns regarding the specificity of these synthetic ligands for further investigation of G4s, especially for rG4 isolation purposes, have been raised. In comparison to G4 ligands, we propose a novel magnetic bead-based pulldown assay that enables the selective capture of general rG4s using functionalized l-Apt.4-1c from both simple buffer and complex media, including total RNA and the cell lysate. We found that our l-RNA aptamer can pulldown general rG4s with a higher efficiency and specificity than the G4 small molecule ligand BioTASQ v.1 in the presence of non-target competitors, including dG4 and non-G4 structures. Our findings reveal that biotinylated l-aptamers can serve as effective molecular tools for the affinity-based enrichment of rG4 of interest using this new assay, which was also verified by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) on endogenous transcripts. This work provides new and important insights into rG4 isolation using a functionalized l-aptamer, which can potentially be applied in a transcript-specific or transcriptome-wide manner in the future.

SUBMITTER: Lam SY 

PROVIDER: S-EPMC11359968 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Capture of RNA G-quadruplex structures using an l-RNA aptamer.

Lam Sin Yu SY   Umar Mubarak Ishaq MI   Zhao Haizhou H   Zhao Jieyu J   Kwok Chun Kit CK  

RSC chemical biology 20240829


G-quadruplexes (dG4 and rG4) are nucleic acid secondary structures formed by the self-assembly of certain G-rich sequences, and they have distinctive chemical properties and play crucial roles in fundamental biological processes. Small molecule G4 ligands were shown to be crucial in characterizing G4s and understanding their functions. Nevertheless, concerns regarding the specificity of these synthetic ligands for further investigation of G4s, especially for rG4 isolation purposes, have been rai  ...[more]

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