Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics

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.

小干扰RNA（siRNA）治疗剂可为降低致病基因的表达提供兼具选择性与强效性的技术路径。此类治疗方式若要获得监管审批，需进行序列确证，而该步骤通常通过完整串联质谱测序完成。然而，该方法会生成极为复杂的质谱谱图，难以解析，且通常无法实现完整的序列覆盖度。本研究旨在开发一种自下而上式siRNA测序平台，以简化测序数据分析流程并实现完整的序列覆盖。与自下而上蛋白质组学类似，该流程需通过化学或酶解消化将寡核苷酸链缩短至可分析的长度，但siRNA通常带有会抑制降解过程的修饰基团。本研究测试了六种消化方案对2'-修饰siRNA的消化可行性，最终确定核酸酶P1可提供高效的消化工作流程。通过部分消化策略，核酸酶P1可实现较高的5'端与3'端序列覆盖度，并生成多种重叠的消化产物。此外，无论RNA的硫代磷酸酯修饰比例、2'-氟修饰状态、序列组成或链长如何，该酶均可实现高质量且高重现性的RNA测序。综上，本研究开发了一种基于核酸酶P1的稳健酶解方案，用于自下而上式siRNA测序，可集成至现有序列确证工作流程中。