Surface Plasmon Resonance Analysis for Evaluating ASO Targeting Structured RNA.

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• Antisense oligonucleotides (ASOs) are crucial for gene expression regulation via sequence-specific RNA hybridization.
• Target RNA higher-order structures significantly impact ASO accessibility and binding kinetics.
• Conventional UV melting analysis often overlooks these critical RNA structural features.

Purpose of the Study:

• To investigate surface plasmon resonance (SPR) as an in vitro method for evaluating ASO binding, considering RNA structural constraints.
• To compare ASO binding to linear RNA fragments versus structured RNA targets.
• To assess SPR's utility as an alternative to UV melting for ASO screening.

Main Methods:

• Designed multiple ASOs targeting the PRF84 RNA motif involved in HIV-1 gag-pol expression.
• Utilized SPR analysis to compare ASO interactions with complementary RNA fragments and the folded PRF84 structure.
• Analyzed differences in ASO binding behavior based on target RNA conformation.

Main Results:

• Identical ASOs demonstrated markedly different binding behaviors when interacting with linear RNA versus the structured PRF84.
• RNA structure was confirmed to be a significant determinant of ASO binding affinity and kinetics.
• SPR analysis revealed distinct interaction profiles influenced by target RNA conformation.

Conclusions:

• SPR analysis provides a valuable method for assessing ASO-RNA interactions that incorporates crucial RNA structural information.
• SPR serves as a potentially superior alternative to UV melting analysis for screening ASOs, especially those targeting structured RNAs.
• This approach enhances the understanding of ASO efficacy by accounting for biological target structure.