Structural characterization of fully chemically modified siRNA with lipid conjugate and divalent architecture
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 development of delivery platforms, including lipid conjugates and multivalent scaffolds, has accelerated the emergence of extrahepatic-targeting small interfering RNA (siRNA). These molecular architectures have advanced multiple clinical-stage siRNA candidates targeting the central nervous system (CNS) and other extrahepatic tissues, underscoring an urgent need for analytical methodologies capable of resolving conjugate-derived fragmentation pathways and symmetric multivalent structures that complicate their characterization by tandem mass spectrometry (MS/MS). Here, we develop complementary MS/MS and data-interpretation strategies for structural characterization of lipid-conjugated and fully chemically modified divalent siRNA using ion-pair-free hydrophilic interaction liquid chromatography (HILIC). Conjugate-derived fragmentation pathways were first established using a lipid-conjugated oligonucleotide standard via collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD), and the resulting framework was then applied to characterize a human Huntingtin (HTT)-targeting lipid-conjugated siRNA. For analysis of a symmetric divalent siRNA, a customized terminal fragment-based data-processing strategy was developed that preserved the complete molecular architecture and enabled assignment of terminal fragment ions from both halves of the construct without relying on internal fragments. This workflow provided comprehensive sequence characterization of divalent siRNA while substantially improving recovery of informative 3ʹ-terminal fragment ions compared with the conventional static 3ʹ-modification method. Collectively, the lipid-fragmentation framework and terminal fragment-based interpretation approach provided a robust analytical platform for structural characterization of increasingly complex conjugated and multivalent RNA therapeutics.
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BibTeX
@misc{gadallah2026structural,
title = {{Structural characterization of fully chemically modified siRNA with lipid conjugate and divalent architecture}},
author = {Mohamed I. Gadallah and Noha M El Zahar and Thanh Nguyen and Kailyn L. Nonhof and Nicholas McHugh and Ken Yamada and Dimas Echeverria and Jennifer S. Brodbelt},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15008847/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008847/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Structural characterization of fully chemically modified siRNA with lipid conjugate and divalent architecture
AU - Mohamed I. Gadallah
AU - Noha M El Zahar
AU - Thanh Nguyen
AU - Kailyn L. Nonhof
AU - Nicholas McHugh
AU - Ken Yamada
AU - Dimas Echeverria
AU - Jennifer S. Brodbelt
PY - 2026
DA - 2026/09/15
PB - ChemRxiv
DO - 10.26434/chemrxiv.15008847/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008847/v1
AB - The development of delivery platforms, including lipid conjugates and multivalent scaffolds, has accelerated the emergence of extrahepatic-targeting small interfering RNA (siRNA). These molecular architectures have advanced multiple clinical-stage siRNA candidates targeting the central nervous system (CNS) and other extrahepatic tissues, underscoring an urgent need for analytical methodologies capable of resolving conjugate-derived fragmentation pathways and symmetric multivalent structures that complicate their characterization by tandem mass spectrometry (MS/MS). Here, we develop complementary MS/MS and data-interpretation strategies for structural characterization of lipid-conjugated and fully chemically modified divalent siRNA using ion-pair-free hydrophilic interaction liquid chromatography (HILIC). Conjugate-derived fragmentation pathways were first established using a lipid-conjugated oligonucleotide standard via collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD), and the resulting framework was then applied to characterize a human Huntingtin (HTT)-targeting lipid-conjugated siRNA. For analysis of a symmetric divalent siRNA, a customized terminal fragment-based data-processing strategy was developed that preserved the complete molecular architecture and enabled assignment of terminal fragment ions from both halves of the construct without relying on internal fragments. This workflow provided comprehensive sequence characterization of divalent siRNA while substantially improving recovery of informative 3ʹ-terminal fragment ions compared with the conventional static 3ʹ-modification method. Collectively, the lipid-fragmentation framework and terminal fragment-based interpretation approach provided a robust analytical platform for structural characterization of increasingly complex conjugated and multivalent RNA therapeutics.
ER -