Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics.
Anal Chem
; 95(9): 4404-4411, 2023 03 07.
Article
em En
| MEDLINE
| ID: mdl-36812429
ABSTRACT
siRNA therapeutics provide a selective and powerful approach to reduce the expression of disease-causing genes. For regulatory approval, these modalities require sequence confirmation which is typically achieved by intact tandem mass spectrometry sequencing. However, this process produces highly complex spectra which are difficult to interpret and typically results in less than full sequence coverage. We sought to develop a bottom-up siRNA sequencing platform to ease sequencing data analysis and provide full sequence coverage. Analogous to bottom-up proteomics, this process requires chemical or enzymatic digestion to reduce the oligonucleotide length down to analyzable lengths, but siRNAs commonly contain modifications that inhibit the degradation process. We tested six digestion schemes for their feasibility to digest the 2' modified siRNAs and identified that nuclease P1 provides an effective digestion workflow. Using a partial digestion, nuclease P1 provides high 5' and 3' end sequence coverage with multiple overlapping digestion products. Additionally, this enzyme provides high-quality and highly reproducible RNA sequencing no matter the RNA phosphorothioate content, 2'-fluorination status, sequence, or length. Overall, we developed a robust enzymatic digestion scheme for bottom-up siRNA sequencing using nuclease P1, which can be implemented into existing sequence confirmation workflows.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Digestão
/
Espectrometria de Massas em Tandem
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos