Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics
Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics
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DOI：
10.1021/acs.analchem.2c04902
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发表时间：
2023-02-22
影响因子：
7.4
通讯作者：
Maloney, Todd D.
中科院分区：
文献类型：
--
作者：
Jones, Joshua D.;Grassmyer, Kathleen T.;Maloney, Todd D.
SiRNA疗法提供了一种选择性和强大的方法来减少致病基因的表达。对于监管机构的批准，这些模式需要序列确认，这通常是通过完整的串联质谱学测序实现的。然而，这一过程产生了高度复杂的谱，这很难解释，通常导致不完全的序列覆盖。我们试图开发一个自下而上的siRNA测序平台，以简化测序数据分析并提供完整的序列覆盖。类似于自下而上的蛋白质组学，这个过程需要化学或酶消化，以将寡核苷酸长度减少到可分析的长度，但siRNA通常包含抑制降解过程的修饰。我们测试了六种消化方案对2‘端修饰的siRNAs进行消化的可行性，发现核酸酶P1提供了一种有效的消化工作流程。使用部分消化，核酸酶P1通过多个重叠的消化产物提供高5‘和3’端序列覆盖率。此外，该酶提供高质量和高度重复性的RNA测序，无论RNA硫代含量、2‘-氟化状态、序列或长度。总体而言，我们开发了一种使用核酸酶P1进行自下而上siRNA测序的健壮的酶消化方案，该方案可以在现有的序列确认工作流程中实现。
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.