Oligonucleotide Quantitative Bioanalysis

At NovaBioAssays we provide services to support bioanalysis of oligonucleotide in various of matrix. Based on the size of the oligonucleotide and the purpose of the project, different analytical methods can be selected. The approaches we apply are fit-to-purpose and can be summarized into follow three major analytical methods: 

1. LC-HRMS or LC-MRM/SRM MS assays 
LC–MS/MS and LC–HRAM (high-resolution accurate-MS) methods have been increasingly used for quantitative bioanalysis of oligonucleotide therapeutics and biomarkers due to their superior specificity, good accuracy, excellent precision/reproducibility, large dynamic range and high throughput. The general features for LC-HRMS or LC-MRM/SRM MS assays for oligonucleotide are:
  • Highest specificity
  • Molecular weight identification
  • Product ion characterization
  • LC Separation
  • High throughput (~5 minutes/sample)
  • Wide dynamic range (1000x)
  • Good sensitivity (< 5 ng/mL LLOQ)
  • No enzymes or special reagents needed
  • Good reproducibility (±15/20% acceptance criteria)

An example of LLOQ peak is shown in Figure 1 for a 20-mer PS-ODN in Human Plasma. A typical separation of two strand RNA oligos of dsRNA are shown in Figure 2.

Figure 1. Typical LLOQ peak (left) of a LC-MS/MS Assay for a 20-mer PS-ODN in Human Plasma

Figure 2. Typical Chromatography Separations of two strand RNA oligos of dsRNA

2. Hybridization based LC-fluorescence assays
Hybridization-based LC-fluorescence assays pair hybridization and LC techniques. The method involves two steps. The target oligonucleotide first hybridizes to a fluorescence-labeled sequence-specific probe oligonucleotide through Watson–Crick base pairs. The resulting fluorescence labeled duplex is then subjected to a strong-anion-exchange HPLC (SAX-HPLC) analysis monitored by a fluorescence detector. Although it is a hybridization-based assay, the LC-fluorescence analysis makes it very different from other hybridization based assays that always involve the use of enzyme(s).  The advantages of this technique are a combination of the merits of traditional hybridization ELISA (high sensitivity and simple sample preparation process) and conventional HPLC-UV/fluorescence assays (high accuracy/precision, broad dynamic range, good reproducibility and high specificity)

3. hELISA assays
We also provide services for hybridization ELISA. In the sandwich hybridization ELISA assay format, the antigen ligand and antibodies in ELISA are replaced with a nucleic acid analyte, complementary oligonucleotide capture and detection probes.