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Track 1: Advances in Discovery, Formulation, and Delivery of New Modalities

Category: Poster Abstract

(W1230-01-01) Development of Physicochemical Profiling Platform for Therapeutic Oligonucleotides IV Evaluation of Compactness of Oligonucleotides for Determining Their Higher-Order Structure

Wednesday, May 15, 2024
12:30 PM – 1:30 PM PT


Purpose: The physicochemical profiling platform protocol for the formulation development of oligonucleotide pharmaceuticals is still under development. As part of a series of the studies aimed to establish the physical property profiling platform for oligonucleotides, we evaluated compactness of oligonucleotides in solutions using capillary gel electrophoresis (CGE) and 1H diffusion ordered nuclear magnetic resonance spectroscopy (DOSY). We attempted to detect changes in apparent molecular size of oligonucleotides originated from difference in high-order structure. Thrombin aptamer (TBA) and modified TBAs (T1, T4) were used as model oligonucleotides.

Methods: TBA and T1 were designed to form guanine-quadruplex (G4) structure, whereas formation of G4 was not expected for T4 (Table 1). The synthesized oligonucleotides were dissolved in water followed by dilution to 0.1 mg/mL or 0.25 mg/mL with PBS and subjected to CGE. The samples were heat-treated at 90°C for 10 min and dTTP (deoxythymidine triphosphate) was added as the internal standard substances (IS) after cooling. P/ACE MDQ Plus with UV or PDA detector (SCIEX) and ssDNA 100-R kit (SCIEX) were used. The samples were injected by pressure or voltage. The separation voltage and detection wavelength were set at 9.0 kV and 254 nm, respectively. DOSY NMR measurements were performed on the AVANCE NEO 500 MHz (Bruker) equipped with CP DCH 500S2 C/H-D-05 Z probe capable of generating gradient field strength of 53 G/cm. NMR samples were prepared at a concentration of 8 mg/mL using PBS (10% D2O), which were incubated at 90°C for 5 min, followed by slow cooling to room temperature.

Results: In the CGE analysis, the detection method, injection method and injection volume were optimized first. Regarding the detection method, baseline was more stable when UV detector was used than that for the PDA detector. Pressure injection was appropriate to apply the sample, as the peak area was not affected by the solvent composition by this procedure, while it was influenced by voltage injection. The concentrations of oligonucleotides and IS were adjusted to achieve clear separation of their peaks. After establishment of the measurement conditions, TBA, T1 and T4 were analyzed in the presence of urea according to the attached instructions of ssDNA 100-R kit. Urea is known to disrupt hydrogen bonding and destabilize higher-order structures of oligonucleotide. The migration time ratio of T1 to IS (Sp/IS) was larger than those for TBA and T4, which was considered to reflect the large molecular weight of T1. In the absence of urea, the Sp/IS ratio of T1 became smaller and the difference from TBA and T4 became small. The Sp/IS ratio for TBA was smaller than that for T4 despite their same molecular weights. Circular dichroism measurements suggested that TBA and T1 formed G4 structure but T4 did not. Thus, it could be speculated that the small Sp/IS ratio obtained by CGE measurement in the absence of urea might reflect the compactness of the oligonucleotide because of formation of the G4 structure. The diffusion coefficient of oligonucleotides was estimated by DOSY measurement (Table 2). Experimental diffusion coefficients for TBA and T4 agreed with expected values. However, that for T1 was larger than expectation, indicating formation of a more compact structure than expected from the molecular weight. As no single methods provide perfect idea on the higher-order structure of oligonucleotides, both CGE and DOSY analyses should be useful for assuming the structure from a viewpoint of the molecular compactness.

Conclusion: The compactness of oligonucleotides, which is influenced by their higher-order structure, was evaluated by comparing the migration time of CGE in the presence and absence of urea. Moreover, DOSY NMR analysis was performed to evaluate their diffusion coefficient, which should reflect compactness of the molecule. Both methods were proved to be helpful for assuring their higher-order structure.

Acknowledgements: This study was conducted as part of Center of Excellence for Pharmaceutical Materials Science (Materials Open Platform for Pharmaceutical Science) led by National Institute for Materials Science. Conflict of Interest: All authors are employees of each affiliation.

Table 1 Base sequences of oligonucleotides used in this study

Table 2 Diffusion coefficients of oligonucleotides determined by DOSY

Figure 1 Sp/IS of oligonucleotides