Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Affinity chromatography is a powerful technique for biomolecule purification.
• RNA aptamers offer unique binding specificities and can be selected for various targets.
• Sephadex, a dextran-based gel, is widely used in chromatography but lacks specific affinity ligands.

Purpose of the Study:

• To isolate and characterize RNA aptamers with high affinity and specificity for Sephadex.
• To evaluate the potential of these aptamers as affinity tags for RNA purification.

Main Methods:

• In vitro selection (SELEX) was employed to generate RNA ligands binding to Sephadex G-100.
• High-affinity aptamers were identified and characterized for their binding properties.
• Specificity was tested against various matrices and related compounds.

Main Results:

• Two groups of Sephadex-binding RNA aptamers were identified, with strong binding to Sephadex G-100.
• Aptamer D8 demonstrated high affinity for dextran B512, suggesting a binding site of multiple alpha-1,6 linked glucose residues.
• Aptamer D8 showed high specificity for Sephadex, with negligible binding to Sepharose, Sephacryl, cellulose, or pustulan.
• Aptamer D8 enabled a 60,000-fold enrichment of RNA from a complex mixture using Sephadex G-100.

Conclusions:

• Novel RNA aptamers with high affinity and specificity for Sephadex have been developed.
• These aptamers can function as effective affinity tags for the rapid purification of RNA and ribonucleoprotein subunits.
• The findings open new avenues for affinity-based purification strategies utilizing Sephadex matrices.