{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Chemistry",
    "University of Texas at Austin",
    "Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy",
    "Assiut University",
    "Ain Shams University",
    "RNA Therapeutics Institute",
    "University of Massachusetts Chan Medical School"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "affs": [
        0,
        1,
        2,
        3
      ],
      "name": "Mohamed I. Gadallah"
    },
    {
      "affs": [
        0,
        1,
        2,
        4
      ],
      "name": "Noha M El Zahar"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Thanh Nguyen"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Michael B. Lanzillotti"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Kailyn L. Nonhof"
    },
    {
      "affs": [
        5,
        6
      ],
      "name": "Nicholas McHugh"
    },
    {
      "affs": [
        5,
        6
      ],
      "name": "Ken Yamada"
    },
    {
      "affs": [
        5,
        6
      ],
      "name": "Dimas Echeverria"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Jennifer S. Brodbelt"
    },
    {
      "name": "Mohamed I Gaber"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/77nxaeatbpkt7.bib",
  "cited_by": 0,
  "doi": "10.26434/chemrxiv.15008560/v1",
  "fulltext_url": null,
  "id": "77nxaeatbpkt7",
  "issns": [],
  "landing_url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008560/v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1607|Mass Spectrometry Techniques and Applications"
    ]
  ],
  "openalex": "W7212083829",
  "pdf_url": "https://chemrxiv.org/doi/pdf/10.26434/chemrxiv.15008560/v1",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-09",
  "publisher": "American Chemical Society (ACS)",
  "ris": "https://research.draconitic.com/paper/77nxaeatbpkt7.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:19Z",
      "native_id": "10.26434/chemrxiv.15008560",
      "source": "chemrxiv",
      "url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008560/v2"
    }
  ],
  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:1607|Mass Spectrometry Techniques and Applications",
      "subfield": "physical"
    }
  ],
  "title": "Advanced HILIC-MS Analysis of Therapeutic siRNAs with Non-Canonical Backbones and Conjugate Scaffolds",
  "title_text": "Advanced HILIC-MS Analysis of Therapeutic siRNAs with Non-Canonical Backbones and Conjugate Scaffolds",
  "type": "preprint",
  "updated_at": "2026-10-30T12:17:19Z",
  "url": "https://research.draconitic.com/paper/77nxaeatbpkt7",
  "venue": "ChemRxiv",
  "versions": []
}