Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Chemical Biology

Background:

• Antisense oligonucleotides (ASOs) typically inhibit gene expression by blocking RNA-binding proteins.
• Existing ASO modifications primarily focus on target binding and nuclease resistance.

Purpose of the Study:

• To introduce a novel technology for recruiting specific proteins to RNA using chemically modified ASOs.
• To explore the potential of 2'-fluoro (2'-F) modified ASOs for targeted protein recruitment and gene modulation.

Main Methods:

• Synthesis and chemical modification of antisense oligonucleotides (ASOs).
• Formation and characterization of RNA-heteroduplexes with modified ASOs.
• Assessment of protein binding to RNA-heteroduplexes, specifically the interleukin enhancer-binding factor 2 and 3 (ILF2/3) complex.
• Analysis of gene expression modulation via alternative splicing in cell culture and in vivo (mice).

Main Results:

• 2'-F modified ASOs, unlike other tested modifications, form heteroduplexes specifically recognized by the ILF2/3 complex.
• Recruitment of ILF2/3 to precursor mRNA by 2'-F ASOs leads to exon skipping in target transcripts.
• This exon skipping was observed in both cell culture and in a mouse model, demonstrating in vivo efficacy.
• The technology offers a new mechanism for controlling gene expression through targeted protein recruitment.

Conclusions:

• Chemically engineered ASOs can be designed to recruit specific protein complexes to RNA targets.
• 2'-F ASO-mediated recruitment of ILF2/3 provides a novel method for modulating alternative splicing and controlling gene expression.
• This technology holds potential for therapeutic applications by enabling precise gene regulation.