Sample Preparation Tip
Level: Basic
Sample Preparation for Oligonucleotides
Oligonucleotides are highly specific therapeutic agents used to treat a wide range of disease states. During any drug development process the pharmacokinetics of the therapeutics must be assessed and this includes oligo therapeutics, however unlike small molecules, oligos present some unique challenges.
The extraction of oligonucleotides from biological matrices can be regarded as looking for the proverbial needle in a haystack. Even relatively simple biological matrices such as serum contain around 70 mg/mL of protein, approximately 2 mg/mL of lipids in the form of lipoproteins, not to mention the significant amount of salts, organic acids, and sugars present. If we are trying to isolate a therapeutic oligonucleotide at pico to microgram/mL quantities, this is no easy task.
Traditional methods for extracting oligonucleotides use a two-step process: (1) liquid-liquid extraction (LLE) is used to extract the oligonucleotides into the aqueous layer, from which these are subsequently (2) isolated using a standard solid phase extraction (SPE) protocol. This entire process is lengthy and difficult to automate. Clarityâ„¢ OTX eliminates the need for LLE, reducing the opportunities for emulsion formation and sample loss, as well as providing a platform which can be automated. Clarity OTX provides a good ready-to-use solution for extraction of oligonucleotides from plasma and serum samples. The generic protocol is shown in Figure 1.
Figure 1
| Sample Pre-Treatment | |||
|---|---|---|---|
| Biological Fluids | Mix sample with Lysis-Loading buffer (1:1 or 1:2) | ||
| Tissue | 1. Perform a proteinase K digestion or homogenization of the tissue. 2. Mix sample with Lysis-Loading buffer (1:1 or 1:2) |
||
| SPE protocol | |||
| Solvent / Buffer | Volumes | ||
| Step | 96 Well Plate | 100 mg | 2 mg |
| Condition | Methanol | 1 mL | 200 µL |
| Equilibrate | 50 mM Ammonium Acetate, pH 5.5
*For tissue samples add 0.5% TX-100, 0.1% cysteine to the above-mentioned buffer |
1 mL | 200 µL |
| Load | Pre-treated sample | Up to 0.5 mL | 100 µL |
| Wash | 50 mM Ammonium Acetate, pH 5.5/ Acetonitrile (50:50) | 3x 1mL | 2-3 x 100 µL |
| Elute | 100mM Ammonium Bicarbonate, (pH 9.5) / Acetonitrile / Tetrahydrofuran (50:40:10) | 1mL | 2 x 75 µL |
Tissue samples can be particularly challenging requiring the addition of digestion enzymes, such as Proteinase K, and the use of ceramic beads for thorough tissue homogenization and cell lysis. Additionally, depending on the complexity of the tissue sample, optimization of the wash and elution steps may be necessary.
Tips for method optimization:
In the case of wash solvents, different organic additives can be explored though always in combination with a buffered solution at pH 5.5, which is critical to retaining the oligonucleotide on to the sorbent as these are bound through an ion exchange mechanism. An increase in organic content or the use of a different organic additive can help sample clean up by more efficiently eluting contaminants retained by hydrophobic interactions. The advantage of using ion exchange for isolation is that stronger wash solvents can be used as long as pH is maintained.
In the case of the elution step, improved recoveries can be obtained by increasing the eluent solution pH and adjusting the mixture of organic additives used, while maintaining a 50:50 proportion of buffer to organic content. Suggestions for wash and elution buffer optimization are summarized in Figure 2. An additional tip to prevent PS to PO oxidation during the elution step is adding TCEP to the elution buffer.
Figure 2
| Step | Modification |
|---|---|
| Wash | 50mM Ammonium acetate pH 5.5: acetonitrile: MeOH (50:40:10 v/v/v) |
| 50mM Ammonium acetate pH 5.5: acetonitrile (50:50 v/v) | |
| 50mM Ammonium acetate pH 5.5: acetonitrile (30:70 v/v) | |
| 50mM Ammonium acetate pH 5.5: acetonitrile: EtOAC (50:40:10 v/v/v) | |
| Elute | 100mM NH4HCO3 (pH 10): Acetonitrile: THF (50:40:10 v/v/v) |
| 100mM NH4HCO3 (pH 10): Acetonitrile: THF (50:25:25 v/v/v) |
Through appropriate optimization of your oligo extraction using Clarity OTX it is possible to efficiently clean up therapeutic oligos and their metabolites from biological samples; effectively removing cell debris (such as proteins, genomic DNA, and lipids), which can significantly interfere with the oligo therapeutics of interest. By removing these contaminants, MS baseline noise is considerably reduced, resulting in easier quantitative bioanalysis.
Clarity OTX User's Guide for Extracting Oligo Therapeutics from Biological Samples
Comprehensive instructional guide for extracting oligo therapeutics from biological samples.
