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DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers use various methods of DNA extraction. While the specifics of different protocols may vary, some general concepts underlie the process of DNA extraction.
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Affinity chromatography is a powerful technique extensively utilized for separating and purifying specific biomolecules from complex mixtures. It capitalizes on the highly selective binding between an analyte and its counterpart, such as antibody-antigen interactions. The counterpart is immobilized on the stationary phase, forming an affinity column. The stationary phase typically consists of solid support, such as agarose or porous glass beads, immobilizing the affinity ligand. The mobile...
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Related Experiment Video

Updated: Jun 12, 2025

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos
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Removal of dsRNA byproducts using affinity chromatography.

Nathaniel E Clark1, Mateusz Kozarski2, Sinem Demirel Asci2

  • 1Repligen, 16 Cavendish CT, Lebanon, NH 03766, USA.

Molecular Therapy. Nucleic Acids
|June 9, 2025
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Summary

A novel dsRNA-specific affinity resin effectively removes immunogenic double-stranded RNA (dsRNA) byproducts from single-stranded RNA (ssRNA). This purification enhances RNA quality and safety for applications like RNA vaccines and therapeutics.

Keywords:
MT: Oligonucleotides: Therapies and ApplicationsRNA vaccinescircular RNAdouble-stranded RNAin vitro transcriptioninnate immunityinterferonmessenger RNAself-amplifying RNAtherapeutic RNA

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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.