An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA exhibits complex secondary and tertiary structures, hindering the development of sequence-specific targeting ligands.
• Double-stranded RNA (dsRNA) segments are common in RNA tertiary structures, presenting a unique binding challenge.
• Existing DNA-binding motifs like bZIP and bHLH are not ideal for dsRNA major groove recognition due to structural constraints.

Purpose of the Study:

• To design and develop novel ligands capable of sequence-specifically targeting double-stranded RNA (dsRNA).
• To investigate structural requirements for targeting dsRNA major grooves by adapting DNA-binding protein motifs.
• To create a generalizable proteomimetic scaffold for dsRNA recognition.

Main Methods:

• Comparative analysis of high-resolution DNA and RNA structures to identify key features for dsRNA binding.
• Rational design of a synthetic crosslinked helix fork (CHF) motif by modifying bHLH protein structures.
• In vitro evaluation of CHF constructs against RNA and DNA hairpins to assess binding specificity.

Main Results:

• The study identified that replacing leucine zipper motifs with synthetic crosslinkers in bHLH proteins enables dsRNA recognition.
• A model bHLH DNA-binding motif was successfully re-engineered into an RNA ligand.
• The rationally designed CHF scaffold demonstrated specificity for dsRNA targets.

Conclusions:

• A new class of proteomimetics, the CHF scaffold, has been developed for targeting dsRNA.
• This represents an encodable platform for achieving sequence-specific recognition of dsRNA.
• The findings overcome limitations in targeting complex RNA structures and open new avenues in RNA-based therapeutics and diagnostics.