Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Small molecules targeting precursor microRNAs (pre-miRNAs) offer a strategy to modulate microRNA biogenesis.
• Rational drug design is often limited by the complex conformational dynamics of RNA molecules.

Purpose of the Study:

• To define the conformational ensemble of pre-miR-377.
• To understand how the small-molecule binder C1 recognizes pre-miR-377.
• To elucidate the structural and dynamic basis of small-molecule recognition at the pre-miR-377 Dicer site.

Main Methods:

• Site-specific nuclear magnetic resonance (NMR) spectroscopy.
• Enhanced-sampling molecular dynamics (MD) simulations.
• NMR-informed MD and binding simulations.

Main Results:

• The Dicer cleavage site of pre-miR-377 features a dynamic adenosine bulge with U25 shuffling between A3 and A4.
• The small-molecule binder C1 recognizes this dynamic region via minor-groove entry.
• C1 binding stabilizes intermediate conformations and shifts the ensemble away from processing-competent states.
• A conformationally locked mutant demonstrated that RNA dynamics, not a static structure, are crucial for C1 binding.

Conclusions:

• Small-molecule recognition of pre-miR-377 is dependent on its conformational ensemble and dynamics.
• Understanding RNA dynamics is critical for the rational design of RNA-targeted therapeutics.
• This study provides insights into the structural basis for modulating microRNA biogenesis using small molecules.