Advanced HILIC-MS Analysis of Therapeutic siRNAs with Non-Canonical Backbones and Conjugate Scaffolds
7 affiliations
- 1Department of Chemistry
- 2University of Texas at Austin
- 3Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy
- 4Assiut University
- 5Ain Shams University
- 6RNA Therapeutics Institute
- 7University of Massachusetts Chan Medical School
Abstract
The evolution of small interfering RNA (siRNA) therapeutics toward increasingly complex chemical architecture has created analytical challenges for structural characterization. Confident sequencing and site-specific localization of modifications by tandem mass spectrometry (MS/MS) remain difficult due to non-canonical backbone chemistry, diverse chemical modifications and attached conjugates, all contributing to heterogeneous fragmentation. We present an ion-pair-free hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS/MS) workflow that enables non-denaturing and denaturing analysis of conjugated, heavily modified siRNA constructs through temperature-controlled separation. Under non-denaturing conditions, duplexes can be evaluated to assess therapeutic integrity and stability, whereas elevated temperatures achieve duplex dissociation, permitting chromatographic separation and MS/MS analysis of sense and antisense strands. The method was benchmarked using multiple siRNA therapeutics of increasing structural complexity, including the unconjugated duplex ATU027, patisiran, a Huntingtin (HTT)-targeting siRNA containing an extended nucleic acid (exNA) backbone, and a GalNAc-conjugated siRNA drug, fitusiran. Complementary activation methods, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD), produced extensive backbone fragmentation and high sequence coverage while also revealing conjugate-specific fragmentation pathways for GalNAc siRNA. Customized Nucleo-SAFARI software supported confident localization of chemical modifications, non-canonical backbone chemistries, and conjugation sites. These results establish temperature-controlled HILIC–MS/MS as a versatile analytical framework for robust structural characterization of complex therapeutic siRNAs.
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BibTeX
@misc{gadallah2026advanced,
title = {{Advanced HILIC-MS Analysis of Therapeutic siRNAs with Non-Canonical Backbones and Conjugate Scaffolds}},
author = {Mohamed I. Gadallah and Noha M El Zahar and Thanh Nguyen and Michael B. Lanzillotti and Kailyn L. Nonhof and Nicholas McHugh and Ken Yamada and Dimas Echeverria and Jennifer S. Brodbelt and Mohamed I Gaber},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15008560/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008560/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Advanced HILIC-MS Analysis of Therapeutic siRNAs with Non-Canonical Backbones and Conjugate Scaffolds
AU - Mohamed I. Gadallah
AU - Noha M El Zahar
AU - Thanh Nguyen
AU - Michael B. Lanzillotti
AU - Kailyn L. Nonhof
AU - Nicholas McHugh
AU - Ken Yamada
AU - Dimas Echeverria
AU - Jennifer S. Brodbelt
AU - Mohamed I Gaber
PY - 2026
DA - 2026/09/09
PB - ChemRxiv
DO - 10.26434/chemrxiv.15008560/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008560/v1
AB - The evolution of small interfering RNA (siRNA) therapeutics toward increasingly complex chemical architecture has created analytical challenges for structural characterization. Confident sequencing and site-specific localization of modifications by tandem mass spectrometry (MS/MS) remain difficult due to non-canonical backbone chemistry, diverse chemical modifications and attached conjugates, all contributing to heterogeneous fragmentation. We present an ion-pair-free hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS/MS) workflow that enables non-denaturing and denaturing analysis of conjugated, heavily modified siRNA constructs through temperature-controlled separation. Under non-denaturing conditions, duplexes can be evaluated to assess therapeutic integrity and stability, whereas elevated temperatures achieve duplex dissociation, permitting chromatographic separation and MS/MS analysis of sense and antisense strands. The method was benchmarked using multiple siRNA therapeutics of increasing structural complexity, including the unconjugated duplex ATU027, patisiran, a Huntingtin (HTT)-targeting siRNA containing an extended nucleic acid (exNA) backbone, and a GalNAc-conjugated siRNA drug, fitusiran. Complementary activation methods, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD), produced extensive backbone fragmentation and high sequence coverage while also revealing conjugate-specific fragmentation pathways for GalNAc siRNA. Customized Nucleo-SAFARI software supported confident localization of chemical modifications, non-canonical backbone chemistries, and conjugation sites. These results establish temperature-controlled HILIC–MS/MS as a versatile analytical framework for robust structural characterization of complex therapeutic siRNAs.
ER -