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What is RNA analysis by nuclease protection?
A nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration.
Oligonucleotides are traditionally used in polymerase chain reaction applications for in vitro gene amplification. However, recently, oligonucleotides are being evaluated for therapeutic applications either by increasing gene expression via splicing intervention (e.g., Nusinersin in Spinal Muscular Atrophy treatment), decreasing gene expression using anti-sense sequences to inhibit transcription or translation (e.g., Mipomersin inhibition of apoB-100 translation in the treatment of Familial Hypercholesterolemia) or in the case of aptamers, inhibiting protein activity (e.g., Pegaptinib inhibition of vascular endothelial growth factor, VEGF, in the treatment of macular degeneration).
Pharmacokinetic studies for oligonucleotide drugs require specific assays. One assay format that has been put to use by PharmOptima’s scientists is the nuclease protection assay (Figure 1). The nuclease protection assay (see Figure) uses a labeled nucleotide sequence complementary to the oligonucleotide drug to capture free drug from solution. Following capture, the binding reaction is treated with S1 single stranded nuclease to remove non-bound capture oligonucleotide, removing non-specific signal.
PharmOptima uses Meso Scale Discovery (MSD) technology for this assay. The MSD platform, while similar to ELISA, uses electrochemiluminescence detection to analyze complex sample matrices for a variety of analytes.
Assay Characteristics
- Sensitivity pg/mL
- Large dynamic range pg/mL through microgram/mL
- Assay reproducibility
- Quick turn around time (2-3 hours) once assay is established
- Minimal matrix effects
The nuclease protection assay uses a labeled nucleotide sequence complementary to the oligonucleotide drug to capture the oligonucleotide drug from solution forming a double stranded sequence. S1 nuclease is an enzyme that degrades single stranded DNA and RNA. In the nuclease protection assay, following the capture step, S1 nuclease is used to degrade any non-double stranded nucleic acid leaving the double stranded captured drug “protected”. Signal from the double stranded protected sequence is then used for detection and quantification of the oligonucleotide drug.
