Oligonucleotide therapeutics are emerging as a promising modality for targeting disease-associated RNAs. Phosphorothioate (PS)-containing oligonucleotides have gained prominence due to their enhanced stability and pharmacodynamic properties. However, current manufacturing practices afford a mixture of Rp and Sp stereoisomers, and this distribution has been linked to changes in product efficacy. Understanding the sensitivity of analytical methods to changes in this quality attribute has therefore become critically important. Here, we used a suite of analytical techniques—ultraviolet (UV) thermal denaturation, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy—to evaluate the PS diastereomer distribution using Tegsedi, a Food and Drug Administration-approved PS-containing antisense oligonucleotide, and with other synthetic inotersen samples having varied PS diastereomer distributions. While UV and CD techniques showed limited sensitivity, NMR excelled in detecting small changes in the PS diastereomer distribution. The univariate metric of 31P integration was shown to be insufficient for this quality metric evaluation; application of principal component analysis to both 1D 31P and 2D 1H,13C spectra revealed distinct PS changes that arose from the different activators used during manufacturing. This comprehensive evaluation highlights the necessity of advanced analytical techniques in ensuring the quality and consistency of PS-containing oligonucleotide therapeutics.
FireA, XuS, MontgomeryMK, et al.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature1998;391:806–811; doi: 10.1038/35888
ZamecnikPC, StephensonML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A1978;75:280–284; doi: 10.1073/PNAS.75.1.280
4.
HofmanCR, CoreyDR. Targeting RNA with synthetic oligonucleotides: Clinical success invites new challenges HHS public access. Cell Chem Biol2024;31:125–138; doi: 10.1016/j.chembiol.2023.09.005
5.
EgliM, ManoharanM. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res2023;51:2529–2573; doi: 10.1093/NAR/GKAD067
6.
VinjamuriBP, PanJ, PengP. A review on commercial oligonucleotide drug products. J Pharm Sci2024;113:1749–1768; doi: 10.1016/j.xphs.2024.04.021
7.
Biopharma PEG. Nucleic acid therapeutics: Approvals and potential blockbusters. Biopharma PEG; 2024. Available from: https://www.biochempeg.com/article/410.html [Last accessed: September29, 2025].
8.
JoraM, ManzC, ThunbergL, et al.The complexities of oligonucleotide therapeutics: Analytical challenges and opportunities within early drug discovery. Bioanalysis2025;17:1415–1419; doi: 10.1080/17576180.2025.2600912
9.
JadhavV, VaishnawA, FitzgeraldK, et al.RNA interference in the era of nucleic acid therapeutics. Nat Biotechnol2024;42:394–405; doi: 10.1038/s41587-023-02105-y
10.
FreierSM, AltmannK-H. The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA: RNA duplexes. Nucleic Acids Res1997;25:4429–4443; doi: 10.1093/nar/25.22.4429
11.
KhvorovaA, WattsJK. The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol2017;35:238–248; doi: 10.1038/nbt.3765
YuD, KandimallaER, RoskeyA, et al.Stereo-enriched phosphorothioate oligodeoxynucleotides: Synthesis, biophysical and biological properties. Bioorg Med Chem2000;8:275–284; doi: 10.1016/S0968-0896(99)00275-8
14.
Kibler-HerzogL, ZonG, UznanskiB, et al.Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers. Nucleic Acids Res1991;19:2979–2986; doi: 10.1093/nar/19.11.2979
15.
BachelinM, HesslerG, KurzG, et al.Structure of a stereoregular phosphorothioate DNA/RNA duplex. Nat Struct Biol1998;5:271–276; doi: 10.1038/nsb0498-271
16.
LanW, HuZ, ShenJ, et al.Structural investigation into physiological DNA phosphorothioate modification. Sci Rep2016;6:25737; doi: 10.1038/srep25737
17.
IwamotoN, ButlerDCD, SvrzikapaN, et al.Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat Biotechnol2017;35:845–851; doi: 10.1038/nbt.3948
18.
JaroszewskiJW, ClausenV, CohenJS, et al.NMR investigations of duplex stability of phosphorothioate and phosphorodithioate DNA analogues modified in both strands. Nucleic Acids Res1996;24:829–834; doi: 10.1093/nar/24.5.829
19.
SakamuriS, EltepuL, LiuD, et al.Impact of phosphorothioate chirality on double-stranded siRNAs: A systematic evaluation of stereopure siRNA designs. Chembiochem2020;21:1304–1308; doi: 10.1002/cbic.201900630
20.
ØstergaardME, De HoyosCL, WanWB, et al.Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides. Nucleic Acids Res2020;48:1691–1700; doi: 10.1093/nar/gkaa031
21.
JahnsH, TanejaN, WilloughbyJLS, et al.Chirality matters: Stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo. Nucleic Acids Res2022;50:1221–1240; doi: 10.1093/nar/gkab544
22.
EbrahimiSB, EiblingMJ, EnglehartSS, et al.Stereochemistry of phosphorothioate linkages impacts the structure and binding affinity of aptamers and DNAzymes. Mol Pharm2025;22:3198–3207; doi: 10.1021/acs.molpharmaceut.5c00117
23.
MonianP, ShivalilaC, LuG, et al.Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides. Nat Biotechnol2022;40:1093–1102; doi: 10.1038/s41587-022-01225-1
24.
TalapJ, ZhaoJ, ShenM, et al.Recent advances in therapeutic nucleic acids and their analytical methods. J Pharm Biomed Anal2021;206:114368; doi: 10.1016/j.jpba.2021.114368
25.
DemelenneA, ServaisAC, CrommenJ, et al.Analytical techniques currently used in the pharmaceutical industry for the quality control of RNA-based therapeutics and ongoing developments. J Chromatogr A2021;1651:462283; doi: 10.1016/j.chroma.2021.462283
26.
RoussisSG, CedilloI, RentelC. Characterizing the diastereoisomeric distribution of phosphorothioate oligonucleotides by metal ion complexation chromatography, in-series reversed phase-strong anion exchange chromatography, and31P NMR. Anal Chem2021;93:16035–16042; doi: 10.1021/acs.analchem.1c03593
27.
SavitzkyA, GolayMJE. Smoothing and differentiation of data by simplified least squares procedures. Anal Chem1964;36:1627–1639; doi: 10.1021/AC60214A047
28.
RangaduraiA, ShiH, XuY, et al.Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting. Proc Natl Acad Sci U S A2022;119:e2112496119; doi: 10.1073/PNAS.2112496119
29.
DelaglioF, GrzesiekS, VuisterGW, et al.NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J Biomol NMR1995;6:277–293; doi: 10.1007/BF00197809
30.
AliM, ManghraniA, SzramowskiM, et al.The use of multiple liquid chromatography methods augmented by phosphorus-31 nuclear magnetic resonance to characterize the diastereomer composition in synthetic oligonucleotides. J Chromatogr A2026;1766:466600; doi: 10.1016/J.CHROMA.2025.466600
GennaV, Iglesias-FernándezJ, Reyes-FraileL, et al.Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides. Nucleic Acids Res2023;51:4713–4725; doi: 10.1093/nar/gkad309
33.
MiyaharaT, NakatsujiH, SugiyamaH. Similarities and differences between RNA and DNA double-helical structures in circular dichroism spectroscopy: A SAC–CI study. J Phys Chem A2016;120:9008–9018; doi: 10.1021/ACS.JPCA.6B08023
34.
VorlíčkováM, KejnovskáI, BednářováK, et al.Circular dichroism spectroscopy of DNA: From duplexes to quadruplexes. Chirality2012;24:691–698; doi: 10.1002/CHIR.22064
35.
KyprJ, KejnovskáI, RenčiukD, et al.Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res2009;37:1713–1725; doi: 10.1093/NAR/GKP026
36.
LarsonNR, TongF, LiZ, et al.Assessment of stereochemical comparability in phosphorothioated oligonucleotides by CD, 31P NMR, and NP1 digestion coupled to LC-MS. Anal Chem2025;97:6059–6066; doi: 10.1021/acs.analchem.4c06140
37.
DinhNN, WinnBC, ArthurKK, et al.Quantitative spectral comparison by weighted spectral difference for protein higher order structure confirmation. Anal Biochem2014;464:60–62; doi: 10.1016/j.ab.2014.07.011
38.
LeeY, GuS, Al-HashimiHM. Insights into the A-C mismatch conformational ensemble in duplex DNA and its role in genetic processes through a structure-based review. J Mol Biol2024;436:168710; doi: 10.1016/j.jmb.2024.168710
39.
KeY, SharmaE, Wayment-SteeleHK, et al.High-throughput DNA melt measurements enable improved models of DNA folding thermodynamics. Nat Commun2025;16:5572; doi: 10.1038/s41467-025-60455-4
40.
YesselmanJD, DennySK, BisariaN, et al.Sequence-dependent RNA helix conformational preferences predictably impact tertiary structure formation. Proc Natl Acad Sci U S A2019;116:16847–16855; doi: 10.1073/pnas.1901530116
Al-HashimiH, SzekelyO, LeeY, et al.Assessing the contribution of rare DNA states to cancer mutational signatures using sequence-specific conformational fingerprinting. Res Sq [Preprint]2025; doi: 10.21203/rs.3.rs-8012102/v1
43.
ManghraniA, RangaduraiAK, SzekelyO, et al.Quantitative and systematic NMR measurements of sequence-dependent A-T hoogsteen dynamics in the DNA double helix. Biochemistry2025;64:1042–1054; doi: 10.1021/acs.biochem.4c00820
44.
GuS, Al-HashimiHM. Direct measurement of 8OG Syn-anti flips in mutagenic 8OG·A and long-range damage-dependent hoogsteen breathing dynamics using 1H CEST NMR. J Phys Chem B2024;128:4087–4096; doi: 10.1021/acs.jpcb.4c00316
45.
SathyamoorthyB, SannapureddiRKR, NegiD, et al.Conformational characterization of duplex DNA with solution-state NMR spectroscopy. J Magn Reson Open2022;10–11:100035; doi: 10.1016/j.jmro.2022.100035
46.
HirschbeinBL, FearonKL. Commentary 31P NMR spectroscopy in oligonucleotide research and development. Antisense Nucleic Acid Drug Dev1997;7:55–61; doi: 10.1089/oli.1.1997.7.55
47.
LiJ, ChenF, ZhangD, et al.An accurate and fast 31P qNMR assay method for oligonucleotide therapeutics. Anal Chem2024;96:16514–16519; doi: 10.1021/acs.analchem.4c03693
48.
BjørstorpS, MalmstrømJ. Quantitative 31P NMR spectroscopy platform method for the assay of oligonucleotides as pure drug substances and in drug product formulations using the internal standard method. Anal Chem2024;96:11198–11204; doi: 10.1021/acs.analchem.4c00419
49.
ArbogastLW, DelaglioF, BrinsonRG, et al.Assessment of the higher-order structure of formulated monoclonal antibody therapeutics by 2D methyl correlated NMR and principal component analysis. Curr Protoc Protein Sci2020;100:e105; doi: 10.1002/cpps.105
50.
BrinsonRG, ElliottKW, ArbogastLW, et al.Principal component analysis for automated classification of 2D spectra and interferograms of protein therapeutics: Influence of noise, reconstruction details, and data preparation. J Biomol NMR2020;74:643–656; doi: 10.1007/s10858-020-00332-y
51.
BrinsonRG, ArbogastLW, MarinoJP, et al.Best practices in utilization of 2D-NMR spectral data as the input for chemometric analysis in biopharmaceutical applications. J Chem Inf Model2020;60:2339–2355; doi: 10.1021/acs.jcim.0c00081
52.
SchaeferC, CornetE, PiottoM. NMR coupled with multivariate data analysis for monitoring the degradation of a formulated therapeutic monoclonal antibody. Int J Pharm2024;667:124894; doi: 10.1016/J.IJPHARM.2024.124894
53.
AbdiH, WilliamsLJ. Principal component analysis. WIREs Computational Stats2010;2:433–459; doi: 10.1002/wics.101
54.
SolomonTL, DelaglioF, GiddensJP, et al.Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb. MAbs2023;15:2160227; doi: 10.1080/19420862.2022.2160227
55.
ArricoL, StolfiC, MarafiniI, et al.Inhomogeneous diastereomeric composition of mongersen antisense phosphorothioate oligonucleotide preparations and related pharmacological activity impairment. Nucleic Acid Ther2022;32:312–320; doi: 10.1089/nat.2021.0089
56.
LovmarL, AhlfordA, JonssonM, et al.Silhouette scores for assessment of SNP genotype clusters. BMC Genomics2005;6:35; doi: 10.1186/1471-2164-6-35
57.
RousseeuwPJ. Silhouettes: A graphical aid to the interpretation and validation of cluster analysis. J Comput Appl Math1987;20:53–65; doi: 10.1016/0377-0427(87)90125-7
58.
Mohana Rao KakitaV, HosurRV. Mahalanobis distance correlation: A novel approach for quantitating changes in multidimensional NMR spectra in biological applications. J Magn Reson2022;337:107165; doi: 10.1016/j.jmr.2022.107165
59.
ChenK, ParkJ, LiF, et al.Chemometric methods to quantify 1D and 2D NMR spectral differences among similar protein therapeutics. AAPS PharmSciTech2018;19:1011–1019; doi: 10.1208/s12249-017-0911-1
60.
BecetteOB, TranA, JonesJW, et al.Structural fingerprinting of siRNA therapeutics by solution NMR spectroscopy. Nucleic Acid Ther2022;32:267–279; doi: 10.1089/nat.2021.0098
61.
RangaduraiA, SzymaskiES, KimseyIJ, et al.Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R1ρ relaxation dispersion. Prog Nucl Magn Reson Spectrosc2019;112–113:55–102; doi: 10.1016/J.PNMRS.2019.05.002
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
