Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET.

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• Understanding RNA folding, interactions, and localization requires advanced transcriptome tools.
• Förster resonance energy transfer (FRET) typically measures distances, assuming rotational averaging, limiting its use for angle-dependent nucleic acid studies.
• Incorporating fluorophores rigidly and biocompatibly into nucleic acid substructures for angle-resolved FRET is challenging.

Purpose of the Study:

• To develop and apply angle-dependent FRET measurements for RNA structural analysis.
• To utilize genetically encodable fluorescent aptamers (Broccoli and Mango-III) for measuring angular FRET.
• To demonstrate the capability of this method in detecting structural changes in RNA, such as riboswitch conformational transitions.

Main Methods:

• Employed fluorescent RNA aptamers Broccoli (donor) and Mango-III (acceptor) for FRET measurements.
• Constructed constructs with a variable-length helix connecting the aptamers to systematically rotate the acceptor relative to the donor.
• Validated FRET oscillation patterns against simulated data from oriented fluorophore models.

Main Results:

• Observed sinusoidal oscillations in FRET efficiency as a function of helix length, confirming angular dependence.
• Demonstrated that the method accurately reflects the orientation of fluorophores separated by an inflexible linker.
• Showcased the structural rigidification of the NiCo riboswitch upon binding transition metal ions using this technique.

Conclusions:

• Fluorescence turn-on aptamers provide a robust platform for angle-resolved FRET measurements in RNA.
• This approach overcomes limitations of traditional FRET by enabling precise orientation determination.
• The method enhances structural interpretation of both ensemble and single-molecule FRET data for RNA studies.