(194d) Ultrafiltration of Ribonucleic Acids

There is a growing interest in the use of nucleic acids, and in particular RNA, as an alternative to conventional therapies in the treatment of AIDS, cancer, and a wide range of genetic disorders. Short interfering (siRNA) and micro (miRNA) RNA can provide unique ability to modulate gene expression. The successful commercialization of these RNA-based therapies requires the development of a cost-effective platform for the purification of the therapeutic RNA, including removal of host cell proteins, DNA, and other nucleic acid fragments. Several recent studies have demonstrated the feasibility of using small pore size ultrafiltration membranes for the purification of supercoiled plasmid DNA, but it is difficult to extrapolate these results to RNA due to differences in both the size and structure of the nucleic acids. The objective of this work was to evaluate the potential of using ultrafiltration for RNA purification.

Ribonucleic acid from Torula yeast was used as a model RNA. The RNA was suspended in Tris-EDTA buffer, with concentrations measured using the Quant-iT RiboGreen RNA Assay. RNA integrity was examined by Agarose Gel Electrophoresis with the gels visualized using GelStar Nucleic Acid Gel Stain. Ultrafiltration experiments were performed in a stirred cell apparatus using Ultracel composite regenerated cellulose membranes with 100 kD nominal molecular weight cutoff (pore size of approximately 10 nm).

RNA transmission increased with increasing filtrate flux, similar to behavior observed previously with plasmid DNA. This is likely due to the elongation of the RNA molecules in the converging flow field entering the membrane pores. However, the RNA transmission decreased with increasing ionic strength, which is exactly the opposite of the behavior observed with DNA. This effect was quite pronounced. For example, at a flux of 0.1 cm/s, the RNA transmission decreased from more than 60% in the TE buffer to less than 2% in the same buffer with 10 mM NaCl. This large effect of ionic strength on RNA transmission is likely due to changes in the secondary and / or tertiary structure of the RNA due to strong intramolecular interactions. These results provide important insights into the possible use of ultrafiltration for RNA purification, including the separation between DNA and RNA under different ionic conditions.