Nucleotide flipping correlates with fluorescence activation in the RhoBAST imaging platform

Area of Science:

• Molecular Biology
• Structural Biology
• Biophysics

Background:

• Super-resolution imaging requires advanced molecular probes for high spatiotemporal resolution.
• Fluorogenic RNA aptamers offer unique advantages for RNA dynamics studies.
• Understanding the photophysical properties of probes like RhoBAST is crucial for optimizing imaging techniques.

Purpose of the Study:

• To determine the structure of the RhoBAST fluorogenic RNA aptamer and its ligand-bound complexes.
• To elucidate the molecular mechanisms behind RhoBAST's exceptional photophysical properties.
• To provide a structural basis for designing improved RNA imaging tools.

Main Methods:

• X-ray crystallography to determine the free-form and ligand-bound structures of RhoBAST.
• Structure-guided mutagenesis to investigate nucleotide function.
• Biophysical assays including fluorescence spectroscopy and surface plasmon resonance.
• 2-aminopurine kinetics to study ligand binding dynamics.

Main Results:

• The first free-form structure of RhoBAST revealed an inverted 'V'-shaped architecture.
• Ligand binding induces a conformational transition of nucleotide G38 from inward-facing to outward-flipped.
• This nucleotide flipping mechanism was confirmed to facilitate rapid ligand turnover and fluorescence flickering.
• Mutagenesis and biophysical assays validated the role of nucleotide flipping in RhoBAST's function.

Conclusions:

• A novel nucleotide-flipping mechanism in a fluorogenic RNA aptamer has been identified.
• The structure and mechanism of RhoBAST provide insights into its super-resolution imaging capabilities.
• These findings offer a blueprint for the rational design of next-generation RNA imaging probes.