Removal of dsRNA byproducts using affinity chromatography

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Double-stranded RNA (dsRNA) are immunogenic byproducts of in vitro transcription.
• Removing dsRNA from single-stranded RNA (ssRNA) is challenging due to similar physical properties.
• Immunogenic dsRNA can trigger unwanted innate immune responses, impacting RNA therapeutics and vaccines.

Purpose of the Study:

• To develop a method for selective removal of dsRNA from ssRNA.
• To assess the impact of dsRNA removal on RNA integrity and immunogenicity.
• To evaluate the efficacy of purified RNA in cellular assays.

Main Methods:

• Development of a dsRNA-specific affinity resin.
• Affinity purification of ssRNA to remove dsRNA contaminants.
• Reporter cell line assay to measure innate immune responses.
• Assessment of RNA integrity and protein expression.

Main Results:

• The affinity resin reduced dsRNA levels by over 100-fold, to 0.00007% w/w.
• RNA integrity was maintained after purification.
• Purified RNA, synthesized with standard nucleotides, showed no inflammatory response.
• Purified RNA resulted in greater protein expression and improved cell health compared to unpurified RNA.

Conclusions:

• dsRNA affinity purification is a facile, scalable, and effective method for removing immunogenic dsRNA byproducts.
• This approach significantly improves the quality and safety of transcribed RNA.
• The purified RNA demonstrates reduced immunogenicity and enhanced functionality, making it suitable for RNA vaccines and therapeutics.