RESUMEN
The goal of this study was to develop a reverse transcription quantitative polymerase chain reaction (RT-qPCR) method for the accurate quantification of chemically modified small interfering RNA (siRNA) including but not restricted to thermally destabilizing modifications such as glycol nucleic acid (GNA). RT-qPCR was found to be superior to mass spectrometry-based siRNA detection in terms of sensitivity and throughput. However, mass spectrometry is still the preferred method when specific metabolite detection is required and is also insensitive to siRNA chemical modifications such as GNA. The RT-qPCR approach can be optimized to take chemical modifications into account and works robustly in different matrices without optimization, unlike mass spectrometry. RT-qPCR and mass spectrometry both have their strengths and weaknesses for the detection of siRNA and must be used appropriately depending on the questions at hand. Considerations such as desired throughput, assay sensitivity, and metabolite identification must be weighed when choosing which methodology to apply.
Asunto(s)
Monitoreo de Drogas/métodos , ARN Interferente Pequeño/farmacocinética , Tratamiento con ARN de Interferencia/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Calibración , Monitoreo de Drogas/instrumentación , Glicoles/química , Humanos , Espectrometría de Masas , Medicina de Precisión/instrumentación , Medicina de Precisión/métodos , ARN Interferente Pequeño/sangre , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Sensibilidad y EspecificidadRESUMEN
Aim: A novel single-stranded deaminated oligonucleotide metabolite resulting from a REVERSIR™ oligonucleotide was discovered and identified in monkey liver after subcutaneous administration. Results & methodology: REVERSIR-A and its metabolites were extracted from biological matrices by solid phase extraction and analyzed using LC coupled with high-resolution MS under negative ionization mode. A novel 9-mer metabolite of REVERSIR-A, resulting from deamination of the 3' terminal 2'-O-methyl-adenosine nucleotide to 2'-O-methyl-inosine, was discovered at significant levels in monkey liver. The metabolite's identity was confirmed by LC-MS/MS. Conclusion: This report describes the first observation of a long-chain deaminated metabolite of a single-stranded REVERSIR oligonucleotide in vivo in monkey liver.
