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

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• G-quadruplexes (G4s), including DNA (dG4) and RNA (rG4) forms, are crucial nucleic acid structures with significant biological roles.
• Small molecule G4 ligands are used for G4 characterization, but their specificity, particularly for rG4 isolation, is a concern.

Purpose of the Study:

• To develop a novel, highly specific method for isolating general RNA G-quadruplexes (rG4s).
• To compare the efficacy of a new aptamer-based pulldown assay against existing small molecule G4 ligands.

Main Methods:

• A magnetic bead-based pulldown assay utilizing a functionalized L-RNA aptamer (l-Apt.4-1c).
• Testing the assay's ability to capture general rG4s from simple buffers, total RNA, and cell lysates.
• Comparative analysis with a small molecule G4 ligand (BioTASQ v.1) in the presence of various competitor nucleic acid structures.
• Validation using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) on endogenous transcripts.

Main Results:

• The L-RNA aptamer pulldown assay selectively captured general rG4s with high efficiency and specificity.
• The aptamer assay outperformed the small molecule ligand BioTASQ v.1 in isolating rG4s amidst non-target competitors (dG4, non-G4 structures).
• Biotinylated L-aptamers proved effective for affinity-based enrichment of rG4s, confirmed by RT-qPCR analysis of endogenous targets.

Conclusions:

• Functionalized L-aptamers are effective molecular tools for a novel magnetic bead-based assay for rG4 isolation.
• This aptamer-based method offers superior specificity and efficiency compared to small molecule ligands for rG4 enrichment.
• The assay has potential applications in future transcript-specific or transcriptome-wide rG4 analysis.