Precipitation is a widely adopted method at the bench scale for the purification of various RNA species. It is based on neutralizing the charge of the phosphate backbone and reducing the solubility of RNA in water, achieved by the addition of salts and/or alcohols. Salts provide monovalent cations that neutralize the negatively charged phosphate backbone of RNA, while alcohols change the dielectric constant of the solvent, enhancing electrostatic attraction between positively charged ions and the phosphate backbone. The end result is the precipitation of RNA, which can be collected by centrifugation and resuspension. This blog will focus solely on the different salt options.
There are several salts that can be used for RNA purification, such as lithium chloride (LiCl), ammonium acetate, and sodium acetate. Below is a summary of applications for the three different types of salts (Lorsch et al., 2013).
- Lithium chloride (LiCl): Efficient at precipitating larger RNA molecules (including mRNA and rRNA) but not DNA, tRNA, small RNA fragments, proteins, and nucleotides (Barlow et al., 1963). This makes it ideal for mRNA or rRNA purification, especially after in vitro transcription (IVT). However, it might be less effective for low concentrations of RNA, for which ethanol and a different salt might be more suitable. This is the most widely used method at bench scale due to its simplicity and specificity.
- Ammonium acetate: Efficient for precipitating both small and large RNAs but does not precipitate nucleotides. It’s a good choice for RNA purification after reactions. However, it also precipitates proteins, so phenol/chloroform extractions are typically performed first.
- Sodium acetate: Highly efficient at precipitating all nucleic acids, including nucleotides, DNA, and small RNA fragments. It doesn’t inhibit many of the reactions often performed with purified RNAs, making it a versatile choice.
Therefore, to purify mRNA, LiCl is the most ideal option. However, why is there specificity for mRNA and rRNA? Currently, there’s no single clear mechanism that is well understood. Normally, it is attributed to the difference in charge when comparing all the different populations. Other than large RNA molecules like mRNA or rRNA, there are three different categories:
- Small molecules including tRNA, small RNA fragments, nucleotides, etc.: These molecules are either nucleotides or made of a chain of nucleotides. The total charge is not strictly proportional to the number of nucleotides since different nucleotides have different charges. But it’s relatively safe to say that the longer the sequence, the larger the overall charge is. Since these are small molecules with a small number of nucleotides, therefore, their negative charge is much smaller compared to large molecules like mRNA or rRNA.
- Proteins are small molecules with different isoelectric points. The isoelectric point (pI) is defined as the pH at which the protein/peptide has a net charge of zero. Proteins have a wide pI range from pH 4-12. At pH higher than pI, proteins are negatively charged; at pH lower than pI, proteins are positively charged. But due to the relatively small size of proteins, the overall charge, even if negative, is quite different from a large RNA molecule.
- DNA is double-stranded molecules. It means that, at the same length, DNA has twice the number of phosphate groups attached compared to mRNA, thus roughly twice the charge of mRNA. The difference in charge can be explored when optimizing conditions for precipitation purification.
However, even though the difference in charge is non-trivial and can partially explain why LiCl, with a very well-controlled concentration, can be used to separate out mRNA/rRNA from the other impurity populations, it couldn’t give a satisfying reasoning as to why the other salts, when explored carefully, can’t achieve the same separation results. It remains to be seen how future research can unveil the exact underlying mechanism of the specificity of LiCl for large RNA molecule precipitation.
Reference:
- Barlow, J. J., et al. “A simple method for the quantitative isolation of undegraded high molecular weight ribonucleic acid.” Biochemical and Biophysical Research Communications 13.1 (1963): 61-66.
- Lorsch, Jon. Laboratory methods in enzymology: RNA. Academic Press, 2013.