(W1030-01-01) Development of Physicochemical Profiling Platform for Therapeutic Oligonucleotides III Thermodynamic Analysis on Higher-Order Structure of Oligonucleotides

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. This study focuses on thermodynamic 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 TBAs were subjected to differential scanning calorimetry (DSC) measurements (MicroCal PEAQ-DSC), where two types of phosphate buffers that mimicked environment inside and outside of cells (PBS_in and PBS_out, respectively) were used at a sample concentration of 0.5 mg/mL. The most important difference between PBS_in and PBS_out are the difference in the KCl concentration (57.3 mM for PBS_in, 2.68 mM for PBS_out). In the measurement, the samples were heated from 5 °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. Then, the samples were heated again to 90 °C at a rate of 1 °C/min.

Results: TBA forms guanine quadruplex (G4) structure which includes two G quartets. T1 to T4 were designed to change the higher-order structure. Figure 2 shows DSC curves of each sample, where effect of thermal history and PBS types were also investigated. The curves of the first and the second heating agreed each other in PBS_in for all samples, whereas those for T2 and T3 changed in PBS_out. The curves for these modified TBAs exhibited multiple peaks during the first heating, suggesting requirement of the heat treatment to obtain homogeneous higher-order structure for these samples. The obtained thermodynamic parameters for the second heating are summarized in Table 2. The melting temperature (T_m) of all samples agreed well with those obtained by UV melting curves except that T_m for T4 was detected only by DSC. The melting enthalpy, ΔH_m, was always larger in PBS_in except for T1. Those for T4 were much smaller than other samples, suggesting that the molecular interactions to form the higher-order structure was weak for T4. Although DG for TBA was negative and positive in PBS_in and PBS_out at 37 °C, respectively, indicating that the equilibrium higher-order structures for TBA are different under these temperature conditions. DH_m value for T1 was about 1.5 times of that for TBA in PBS_in, and that for T2 was between them, suggesting that the structuring and melting behaviors are dominated by the G4 structure, whereas intermolecular interaction may partially be responsible in PBS_out. The DSC investigation revealed the relationship between base sequences and thermodynamic stability of oligonucleotides.

Conclusion: The thermodynamic behavior of TBA and modified TBAs were investigated by DSC. Increase in number of G quartets raised the melting temperature. The melting enthalpy was roughly proportional to number of G4 quartets, suggesting that G4 quartets are responsible for the melting behavior.

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. The authors declare no conflict of interest.

Table 1. TBA and Modified TBAs used in this study.

Figure 1. DSC curves of oligonucleotides: The first heating (blue), cooling (green), and the second heating (red) in PBSin (left) and PBSout (right).


Table 2. Thermodynamic parameters for melting of TBA, T1, T2, T3 and T4 in PBSin and PBSout.