Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Structural Fingerprinting of Antisense Oligonucleotide Therapeutics by Solution NMR Spectroscopy



Owen Becette, John Marino, Robert Brinson


Purpose. Antisense oligonucleotide (ASO) therapeutics are an emerging class of biopharmaceuticals to treat and prevent diseases, particularly those involving "undruggable" protein targets. Impurities generated throughout the ASO drug manufacturing and formulation pipeline can be detrimental to drug safety and efficacy. Therefore, analytical techniques are needed to rigorously characterize these molecules for quality assurance purposes. Methods. We demonstrate 1D and 2D nuclear magnetic resonance (NMR) spectroscopy methods that can generate highresolution structural "fingerprints" of ASOs. Results and Conclusions. 1D 1H and 31P measurements are shown to provide rapid initial assessment of the ASO integrity. In particular, a well-resolved pair of 31P signals arising from the 5´-end of the phosphorodiamidate morpholino oligomer (PMO) are sensitive to complex formation and oligomerization state. 2D 1H-1H,1H-13C, and 1H-15 N experiments, although less sensitive, are further shown to enable resonance assignment, which will allow the tracking of structural changes at high-resolution during the drug development and manufacturing processes. We further anticipate that the described NMR approaches will be broadly applicable to fully formulated ASO therapeutics, including modalities other than PMOs.
Nucleic Acid Therapeutics


NMR fingerprinting, oligonucleotide therapeutics, drug development, structure, quality attributes, ASO


Becette, O. , Marino, J. and Brinson, R. (2022), Structural Fingerprinting of Antisense Oligonucleotide Therapeutics by Solution NMR Spectroscopy, Nucleic Acid Therapeutics, [online],, (Accessed May 23, 2024)


If you have any questions about this publication or are having problems accessing it, please contact

Created October 4, 2022, Updated February 27, 2023