Bimodal loop-loop interactions increase the affinity of RNA aptamers for HIV-1 RNA structures

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Therapeutics

Background:

• RNA molecules play crucial roles in gene regulation through interactions like loop-loop binding between adjacent hairpins.
• Natural RNA interactions can be mimicked to design novel RNA ligands with specific binding capabilities.

Purpose of the Study:

• To design and validate a double kissing complex model for creating bivalent RNA ligands capable of simultaneously recognizing at least two structured RNA targets.
• To investigate the enhanced binding affinity of these bivalent RNA ligands compared to individual aptamers.

Main Methods:

• In vitro selection was used to identify hairpin aptamers.
• A double kissing complex model was designed using two HIV-1 transactivating responsive (TAR) RNA variants (BRU and MAL).
• Binding interactions were analyzed using thermal denaturation (UV spectroscopy), electrophoretic mobility shift assays (EMSAs), and surface plasmon resonance (SPR).

Main Results:

• The bimodal complex exhibited a binding equilibrium constant at least one order of magnitude higher than individual hairpin complexes, primarily due to a slower dissociation rate.
• Bivalent RNA ligands targeting functional motifs in the HIV-1 5' untranslated region (5'UTR) also showed enhanced target affinity.
• Specifically, ligands targeting TAR and dimerization initiation site (DIS) or TAR and poly(A) hairpins demonstrated superior binding.

Conclusions:

• Bimodal structured RNA ligands can effectively mimic natural RNA interactions to achieve simultaneous recognition of multiple RNA targets.
• This strategy significantly enhances the binding affinity of RNA aptamers for their targets.
• Bivalent RNA ligands represent a promising approach for developing high-affinity aptamers for therapeutic and diagnostic applications targeting structured RNA motifs.