Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Therapeutic small interfering RNA (siRNA) drug development is rapidly expanding.
• siRNA functions by silencing target gene expression via the RNA-induced silencing complex (RISC).
• The mechanism of RISC loading, particularly the role of double-stranded siRNA, requires further clarification.

Purpose of the Study:

• To investigate the loading of double-stranded siRNA into RISC.
• To develop a novel assay for accurate quantification of RISC-loaded siRNA.
• To improve the specificity and accuracy of pharmacokinetic assessments for siRNA therapeutics.

Main Methods:

• RNA immunoprecipitation combined with a probe-based hybridization liquid chromatography-fluorescence assay.
• Quantification of RISC-bound antisense strands, distinguishing between double-stranded and single-stranded forms.
• Discrimination between 5'-phosphorylated antisense (5'p-AS) and nonphosphorylated forms of RISC-bound siRNA.

Main Results:

• Demonstrated that double-stranded siRNA can load into RISC.
• Developed a novel assay that accurately quantifies RISC-bound antisense strands irrespective of their duplex or single-stranded state.
• The assay specifically quantifies the 5'p-AS form and outperforms stem-loop qPCR for mixed siRNA forms.

Conclusions:

• Double-stranded siRNA loading into RISC offers new insights into RISC loading mechanisms.
• The developed probe-hybridization LC-fluorescence assay provides a more accurate and specific method for quantifying RISC-associated siRNA.
• This technique is crucial for reliable pharmacokinetic assessment of siRNA-based therapeutics.