(W1030-02-09) Characterization of Cell-Penetrating Peptide-Phosphorodiamidate Morpholino Oligonucleotides Using an On-Line Nanoflow Desalting Method

Purpose: Peptide-oligonucleotide conjugate (POCs) are synthetic molecules composed of one or more peptides covalently linked to an oligonucleotide¹. In recent years, POCs have emerged as promising candidates for antisense therapy due to their enhanced cellular uptake and bioavailability. Among these, peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugates have shown exceptional potential. PPMOs consist of a cell-penetrating peptide conjugated to a PMO via an amide linkage. This design significantly improves central nervous system delivery and prolongs median survival compared to unconjugated antisense oligonucleotides for the treatment of spinal muscular atrophy (SMA)². Thus, accurate structural characterization of PPMOs is critical for assessing their chemical integrity, verifying conjugation sites, and identifying sequence variations or modifications that may influence their therapeutic performance. However, this task presents analytical challenges due to the neutral PMO backbone and the heterogeneity introduced by PMO impurities from the synthesis process. To overcome these obstacles, we integrated a high throughput nanoflow on-line desalting method³ coupled with various tandem mass spectrometry (MS/MS) techniques for characterization of PPMOs, demonstrating MS analysis as a robust method for these molecules.

Methods: Standard POCs (6 – 6.5 kDa) were synthesized by reductive amination between oligonucleotides containing 5-formyl-cytosine and lysine-containing peptides. PPMOs (10 – 11 kDa) were synthesized by the Moulton lab. Samples were diluted to 10 µM in 50% ACN or 80% ACN. Online nanoflow desalting was performed using 10 mM piperidine in water on a Dionex RSLCnano Ultimate 3000 LC system. A 5 cm (100 µm ID) fused silica column was packed in-house with Waters Xbridge Phenyl (3.5 µm, 130 Å) pH-resistant stationary phase. All spectra were collected on a Thermo Scientific Orbitrap Lumos Tribrid mass spectrometer modified with a 193 nm excimer laser for ultraviolet photodissociation (UVPD) (1 pulse, 2 mJ). Collision-induced dissociation (CID) was performed with 15-20 normalized collision energy (NCE) using a low q value of 0.14. Electron-based activation method was implemented, including ETD with a 10 ms activation time. For EThcD, ETD was first used for charge reduction, followed by HCD with a NCE of 15. For electrospray ionization mass spectrometry (ESI-MS) experiments, solutions were infused by Au/Pd metal-coated borosilicate emitters with an applied voltage of 0.6 kV. Spectra were collected in both negative and positive mode. Data was interpreted using Nucleo-SAFARI and Prosight Lite.

Results: The nanoflow on-line desalting platform, utilizing an isocratic piperidine-based mobile phase, successfully minimizes salt adduction, increases signal quality, and yields cleaner mass spectra for identification of POCs. During the desalting process, a heterogeneous sample mixture travels through the column while salt adducts are retained by the stationary phase. Nucleic acid analytes are efficiently eluted for further analysis, with the retained salts diverted to waste. High-resolution MS/MS was performed on selected precursor ions of oligonucleotides, providing detailed structural characterization of POCs. UVPD and CID enabled high sequence coverage of both oligonucleotide and peptide portions of standard POCs. CID allowed localization of the 5’-formylcytosine (5fC) at the fifth residue of oligonucleotide portion, while UVPD provided charge-reduced precursor ions and diverse fragment ions, including diagnostic a-B and d/w ions. Moreover, fragment ions at the fifth cytosine on the oligonucleotide and the third lysine on the peptide allow precise confirmation of the conjugation sites on both portions. Overall, sequence coverages of the oligonucleotide and peptide portions were 81% and 90%, respectively. Building upon the success of this workflow, we next investigated more complex therapeutic PPMO conjugates. Prior to implementing the on-line desalting method, we characterized the PPMO conjugates using direct ESI-MS in positive mode. Unlike traditional oligonucleotides, PMOs possess a neutral backbone, while the peptide portion contributes positive charge, facilitating efficient ionization and detection. Charge states from 7+ to 16+ were observed. UVPD provided 82–95% peptide sequence coverage, including a diagnostic z-ion at position 21, confirming conjugation to the PMO. ETD and EThcD yielded complementary fragmentation (50–82% coverage), dominated by c-ions. Together, these results validate our on-line desalting MS/MS workflow for standard POCs and establish a strong foundation for applying the same approach to therapeutic PPMOs.

Conclusion: We demonstrate a robust, high-throughput nanoflow on-line desalting method coupled with MS/MS techniques for detailed characterization of therapeutic PPMOs. Our platform offers enhanced sensitivity, structural coverage, and the ability to resolve complex PPMOs, paving the way for more precise quality control and development of antisense therapeutics.

References: (1) Klabenkova, K.; Fokina, A.; Stetsenko, D. Chemistry of Peptide-Oligonucleotide Conjugates: A Review. Molecules 2021, 26 (17), 5420.
(2) Aslesh, T.; Erkut, E.; Ren, J.; Lim, K. R. Q.; Woo, S.; Hatlevig, S.; Moulton, H. M.; Gosgnach, S.; Greer, J.; Maruyama, R.; Yokota, T. DG9-Conjugated Morpholino Rescues Phenotype in SMA Mice by Reaching the CNS via a Subcutaneous Administration. JCI Insight 2023, 8 (5).
(3) Lanzillotti, M. B.; Brodbelt, J. S. A High-Throughput Workflow for Mass Spectrometry Analysis of Nucleic Acids by Nanoflow Desalting. Anal. Chem. 2024, 96 (7), 3131–3136.