(T1330-01-01) Development of Physicochemical Profiling Platform for Therapeutic Oligonucleotides II Circular Dichroism Spectroscopy and Nuclear Magnetic Resonance Analysis for Determining Higher-Order Structure

Purpose: Although nucleic acids, which include oligonucleotides, are one of the important classes of new modality drugs, their physicochemical profiling protocol is still under development. We aim to establish the harmonized physicochemical profiling platform for oligonucleotides to achieve their efficient development. Thus, this study focuses on evaluation of the higher-order structure of oligonucleotides using thrombin aptamer (TBA) and modified TBA as model compounds.

Methods: Four modified TBAs (T1 to T4) were designed to control intra/intermolecular interactions to control their higher-order structure (Table 1). TBA and four modified TBA were subjected to Circular Dichroism (CD) and ¹H Nuclear Magnetic Resonance (NMR) measurements. Two types of phosphate buffers that mimic environment inside and outside of cells (PBS_in and PBS_out, respectively) were used for the CD analysis at a sample concentration of 0.2 mg/mL. In the measurement, the samples were heated from 20 °C to 90 °C at a rate of 1 °C/min, followed by incubation at 90 °C and cooling to 5 °C at a rate of 1 °C/min. After incubation at 5 °C for 10 min, the samples were heated again to 90 °C at a rate of 1 °C/min. The spectrum was collected during the heating. In the NMR measurement, samples were dissolved at a concentration of 8 mg/mL using PBS_out (10% D₂O), which was incubated at 90 °C for 5 min, followed by slow cooling to room temperature.

Results: TBA forms guanine quadruplex (G4) structure which consists of stacked two planer G tetrads and four other sequence analogues. T1 to T4 were designed to change the higher-order structure. In the CD analysis, the heat treatment was applied to clarify its necessity, and the topologies identified after the heat treatment are summarized in Table 1. The spectra obtained at 20 and 50 °C during the heating are presented in Figure 1. TBA was proved to form the antiparallel G4 structure, which was characterized by a positive peak at 295 nm and a negative peak at 270 nm in the spectrum. The similar bands were observed for T2 and T3, indicating these modified TBAs also formed the antiparallel G4 structure. However, the band intensity for T2 and T3 was weaker compared to that of TBA, suggesting presence of multiple topologies. CD spectrum for T1 in PBS_in changed during the heat treatment to show the negative band near 240 nm and the positive band near 260 nm after the treatment, which could be assigned as those of the parallel G4 structure. In addition, CD spectrum for T1 in PBS_out displayed the negative band near 240 nm and the positive band near 295 nm. Only very weak peaks were observed in the CD spectra for T4, suggesting absence of the higher-order structure. Except for T4, all modified TBAs showed strong intensity for the positive and negative bands in PBS_in at 50 °C during the 2^nd heating, indicating the importance of potassium for forming the G4 structure. NMR measurements (Figure 2) revealed that TBA including other modified TBAs exhibited the imino proton signals characteristic to the G4 structure except for T4. The number of imino proton peaks of guanines were larger than that of guanine residues in the sequence for TBA, T2 and T3, indicating presence of multiple higher-order structures. These results agree with those obtained by the CD analysis.

Conclusion: The topologies of TBA and modified TBAs were clarified by CD and NMR measurements with information on effect of the thermal history and buffer types. CD was proved to work for clarifying the higher-order structure of the oligonucleotides only with a small amount of the sample, which should be employed in the early stage of the physicochemical profiling study. NMR played a supportive role to clarify presence of the multiple topologies, although larger amount of the sample was required

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

Figure 1

Figure 2