Structural Fingerprinting of Antisense Oligonucleotide Therapeutics by Solution NMR Spectroscopy.

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 high-resolution 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.

Synthesis of an Atom-Specifically 2 H/13 C-Labeled Uridine Ribonucleoside Phosphoramidite.

A combined enzymatic and chemical synthesis of a 2'-O-cyanoethoxymethyl (CEM) protected [1',6-13 C2 , 5-2 H]-uridine phosphoramidite is described herein. This is the first report of an atom-specific nucleobase and ribose labeled 2'-O-CEM protected ribonucleoside phosphoramidite. Importantly, the CEM 2'-OH protecting group permits the efficient solid-phase synthesis of large (>60 nucleotides) RNAs with good yield and purity. The new isotope-labeled phosphoramidite can therefore be applied to nuclear magnetic resonance (NMR) spectroscopy studies. Specifically, the [1',6-13 C2 , 5-2 H]-uridine phosphoramidite can be used to make position-specifically labeled RNAs for NMR analysis without complications from resonance overlap and scalar and dipolar couplings. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the ribonucleoside 6 Basic Protocol 2: Synthesis of the ribonucleoside phosphoramidite 11.

Structural Fingerprinting of siRNA Therapeutics by Solution NMR Spectroscopy.

Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat a wide variety of illnesses, including diseases where traditional drug development efforts have failed. To date, there are 14 short oligonucleotide therapeutics and 2 messenger RNA (mRNA) vaccines approved by the U.S. Food and Drug Administration (FDA), which demonstrates the potential of nucleic acids as a platform for the development of safe and effective medicines and vaccines. Despite the increasing popularity of nucleic acid-based drugs, there has been a paucity of high-resolution structural techniques applied to rigorously characterize these molecules during drug development. Here, we present application of nuclear magnetic resonance (NMR) methods to structurally "fingerprint" short oligonucleotide therapeutics at natural isotope abundance under full formulation conditions. The NMR methods described herein leverage signals arising from the native structural features of nucleic acids, including imino, aromatic, and ribose resonances, in addition to non-native chemistries, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and phosphorothioate (PS) modifications, introduced during drug development. We demonstrate the utility of the NMR methods to structurally "fingerprint" a model short interfering RNA (siRNA) and a sample that simulated the drug product Givosiran. We anticipate broad applicability of the NMR methods to other nucleic acid-based therapeutics due to the generalized nature of the approach and ability to monitor many quality attributes simultaneously.

Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs.

RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce 19F-13C spin pairs into RNA by using a 5-fluorouridine-5'-triphosphate and T7 RNA polymerase-based in vitro transcription. Incorporating the 19F-13C label in two model RNAs produces linewidths that are twice as sharp as the commonly used 1H-13C spin pair. Furthermore, the high sensitivity of the 19F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the 19F-13C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.

Deleterious effects of carbon-carbon dipolar coupling on RNA NMR dynamics.

Many regulatory RNAs undergo dynamic exchanges that are crucial for their biological functions and NMR spectroscopy is a versatile tool for monitoring dynamic motions of biomolecules. Meaningful information on biomolecular dynamics requires an accurate measurement of relaxation parameters such as longitudinal (R1) rates, transverse (R2) rates and heteronuclear Overhauser effect (hNOE). However, earlier studies have shown that the large 13C-13C interactions complicate analysis of the carbon relaxation parameters. To investigate the effect of 13C-13C interactions on RNA dynamic studies, we performed relaxation measurements on various RNA samples with different labeling patterns and compared these measurements with the computational simulations. For uniformly labeled samples, contributions of the neighboring carbon to R1 measurements were observed. These contributions increased with increasing magnetic field and overall correlation time ([Formula: see text]) for R1 rates, necessitating more careful analysis for uniformly labeled large RNAs. In addition, the hNOE measurements were also affected by the adjacent carbon nuclei. Unlike R1 rates, R1ρ rates showed relatively good agreement between uniformly- and site-selectively labeled samples, suggesting no dramatic effect from their attached carbon, in agreement with previous observations. Overall, having more accurate rate measurements avoids complex analysis and will be a key for interpreting 13C relaxation rates for molecular motion that can provide valuable insights into cellular molecular recognition events.

Solid-Phase Chemical Synthesis of Stable Isotope-Labeled RNA to Aid Structure and Dynamics Studies by NMR Spectroscopy.

RNA structure and dynamic studies by NMR spectroscopy suffer from chemical shift overlap and line broadening, both of which become worse as RNA size increases. Incorporation of stable isotope labels into RNA has provided several solutions to these limitations. Nevertheless, the only method to circumvent the problem of spectral overlap completely is the solid-phase chemical synthesis of RNA with labeled RNA phosphoramidites. In this review, we summarize the practical aspects of this methodology for NMR spectroscopy studies of RNA. These types of investigations lie at the intersection of chemistry and biophysics and highlight the need for collaborative efforts to tackle the integrative structural biology problems that exist in the RNA world. Finally, examples of RNA structure and dynamic studies using labeled phosphoramidites are highlighted.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.