Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA

Application of a Ultra Performance Liquid Chromatography Method in the Determination of DNA Quality and Stability

“…This approach was supported by previous studies employing increased column temperatures to separate siRNA. 28 As shown in Figure 2, increased column temperature increases the separation of the linear isoform from the supercoiled isoform, with minimal effects on the open-circle isoform. A plot of retention times at different temperatures for each isoform reveals that retention times of the supercoiled and opencircle forms remain constant with temperature, but the retention time of the linear form increases dramatically above 40 • C, which results in the enhanced separation at elevated temperatures (Fig.…”

“…23,24 However, clear separation of the linear from the supercoiled isoform can create inaccuracies when determining supercoil content. McCarthy et al 28 have reported improved separation of oligonucleotides by utilizing elevated column temperatures, and this work provided the impetus for us to explore a similar strategy for plasmids. As shown in Figures 2 and 3, higher column temperatures resulted in an abrupt increase in the retention time of the linear isoform at 50 • C, which permitted accurate and independent quantification of the supercoiled, open-circle, and linear isoforms.…”

Mass Spectrometry of siRNA

“…In order to properly characterize the state of a siRNA, it is important to maintain the duplex during sample extraction and have a clear methodology to discern minor changes in each strand due to exterior factors, for example, synthesis impurities, storage degradation, or metabolite products. Chromatographic separation of duplex from such truncated duplex species has been shown to be possible, yet difficult [33]. Unfortunately, liquid chromatography-mass spectrometry (LC-MS) analysis of the intact duplex has demonstrated a decrease in sensitivity compared with the denatured individual strands due to increased cation adduction [34].…”

“…Ion-pairing reagents with increasing hydrophobicity such as dimethylbutlyamine (DMBA) [68] and hexylamine (HA) [33] have also been successfully reported in the LC-MS analysis of oligonucleotides and siRNA. They increase HPLC performance by greater oligonucleotide retention and separation at lower ion-pairing concentrations but may be detrimental to ESI signal intensities at concentrations >25 mM.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…A novel mechanism for regulation of gene expression has, for example, recently been discovered, involving the ribonucleic acid interference (RNAi). The RNAi mechanism uses two forms of small RNA molecules, micro RNA which prevents protein synthesis, and small interfering RNA which degrades messenger RNA (McCarthy et al 2009).…”