Improving Oligonucleotide Isolation
To overcome the limitations of two-step isolation, a new methodology was introduced last year for separating oligonucleotides from serum and plasma. This methodology, which has been incorporated in Phenomenex’ Clarity OTX oligonucleotide isolation kit, uses a novel solubilization buffer instead of the time-consuming LLE steps from other protocols.
This method also uses a mixed-mode SPE sorbent for specifically capturing oligonucleotides while allowing the elution of proteins and lipids that can interfere with LC/MS analysis. Being an SPE-based protocol, the isolation methodology is rapid and easily performed in less than 15 minutes.
The Clarity OTX method uses buffers that maintain near-neutral pH throughout the process to avoid unwanted modifications of the oligonucleotide (depurination for DNA below pH 5 and 2´ to 3´ isomerization for RNA above pH 8). The clean-up and recovery of this protocol is demonstrated in Figure 1.
In this example, a 27 mer DNA oligonucleotide was spiked into plasma at the 12 µg/mL level and isolated using the Clarity OTX protocol for analysis by HPLC. The upper HPLC chromatogram of Figure 1 is the plasma-extracted oligonucleotide; the lower chromatogram is the same oligonucleotide directly injected onto the HPLC system. Note the minimum number of UV-interfering contaminants in the plasma-extracted sample.
Because most matrix interference peaks elute away from the oligonucleotide, quantitation is unaffected by matrix interference. Recoveries of greater than 95% from the plasma samples demonstrate the utility of this protocol for isolating oligonucleotides from biological samples. While the data is not shown here, the new isolation method produces a linear response curve with sensitivity down to low nanomolar concentrations, depending on the LC/MS system used.