Toward the Establishment of a Harmonized Physicochemical Profiling Platform for Therapeutic Oligonucleotides: A Case Study for Aptamers Where the Higher-Order Structure Influences Physical Properties.

Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.

Development of RNA/DNA Hydrogel Targeting Toll-Like Receptor 7/8 for Sustained RNA Release and Potent Immune Activation.

Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is an agonist of Toll-like receptor (TLR) 7 and TLR8 and induces strong immune responses. A nanostructured GU-rich RNA/DNA assembly prepared using DNA nanotechnology can be used as an adjuvant capable of improving the biological stability of RNA and promoting efficient RNA delivery to target immune cells. To achieve a sustained supply of GU-rich RNA to immune cells, we developed a GU-rich RNA/DNA hydrogel (RDgel) using nanostructured GU-rich RNA/DNA assembly, from which GU-rich RNA can be released in a sustained manner. A hexapod-like GU-rich RNA/DNA nanostructure, or hexapodRD6, was designed using a 20-mer phosphorothioate-stabilized GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6 were mixed to obtain RDgel. Under serum-containing conditions, GU-rich RNA was gradually released from the RDgel. Fluorescently labeled GU-rich RNA was efficiently taken up by DC2.4 murine dendritic cells and induced a high level of tumor necrosis factor-α release from these cells when it was incorporated into RDgel. These results indicate that the RDgel constructed using DNA nanotechnology can be a useful adjuvant in cancer therapy with sustained RNA release and high immunostimulatory activity.

Development of a Nanostructured RNA/DNA Assembly as an Adjuvant Targeting Toll-Like Receptor 7/8.

Adjuvants are essential for efficiently inducing an antigen-specific immune response in vaccine therapy. Single-stranded RNA (ssRNA) containing guanosine- and uridine-rich sequences is recognized by Toll-like receptor (TLR)7 and/or TLR8 and induces strong immune responses; thus, the application of ssRNA as an adjuvant is desirable. The development of a ssRNA-based adjuvant, however, requires the efficient delivery of ssRNA into the endosomes of antigen-presenting cells, where the TLRs exist. To achieve this, we developed a nanostructured RNA/DNA assembly using DNA nanotechnology, which can be efficiently recognized by antigen-presenting cells. The nanostructured RNA/DNA assembly, named tetrapodRD3, was designed using a 40-mer phosphorothioate-stabilized RNA and three 40-mer phosphodiester DNAs. TetrapodRD3 was more stable than ssRNA under serum conditions. The secreted alkaline phosphatase assay using HEK-Blue hTLR cells showed that tetrapodRD3 triggered human TLR8-specific responses. Fluorescently labeled tetrapodRD3 was efficiently taken up by murine dendritic DC2.4 cells and induced a high level of tumor necrosis factor-α release from the cells. Antigen presentation by the major histocompatibility complex class I on bone marrow-derived dendritic cells was significantly increased by the addition of an antigen along with tetrapodRD3. These results indicate that tetrapodRD3 constructed using DNA nanotechnology can be a useful adjuvant targeting human TLR8.

Application of diffusion ordered-1H-nuclear magnetic resonance spectroscopy to quantify sucrose in beverages.

This work focuses on a quantitative analysis of sucrose using diffusion ordered-quantitative (1)H-nuclear magnetic resonance spectroscopy (DOSY-qNMR), where an analyte can be isolated from interference based on its characteristic diffusion coefficient (D) in gradient magnetic fields. The D value of sucrose in deuterium oxide at 30°C was 4.9 × 10(-10)m(2)/s at field gradient pulse from 5.0 × 10(-2) to 3.0 × 10(-1)T/m, separated from other carbohydrates (glucose and fructose). Good linearity (r(2)=0.9999) was obtained between sucrose (0.5-20.0 g/L) and the resonance area of target glucopyranosyl-α-C1 proton normalised to that of cellobiose C1 proton (100.0 g/L, as an internal standard) in 1D sliced DOSY spectrum. The DOSY-qNMR method was successfully applied to quantify sucrose in orange juice (36.1 ± 0.5 g/L), pineapple juice (53.5 ± 1.1g/L) and a sports drink (24.7 ± 0.6g/L), in good agreement with the results obtained by an F-kit method.

Quantitative analysis of D-(+)-glucose in fruit juices using diffusion ordered-1H nuclear magnetic resonance spectroscopy.

This study works on D-(+)-glucose quantitative analysis using diffusion ordered-quantitative (1)H nuclear magnetic resonance spectroscopy (DOSY-qNMR), by which an analyte could be distinguished from interferences based upon a characteristic diffusion coefficient (D) in gradient magnetic fields. The D value of D-(+)-glucose in deuterium oxide at 30°C was 5.6 × 10(-10) m(2)/s at a field gradient pulse of between 5.0 × 10(-2) and 3.0 × 10(-1) T/m, distinguished from fructose, sucrose and starch. Good linearity (r(2) = 0.9998) was obtained between D-(+)-glucose (0.5-20.0 g/L) and the ratio of the resonance area of α-C1 proton (5.21 ppm) in D-(+)-glucose to that of the ß-C1 proton (5.25 ppm) in D-glucuronic acid (50.0 g/L) as an internal standard. The DOSY-qNMR method was successfully applied to quantify D-(+)-glucose in orange juice (18.3 ± 1.0 g/L), apple juice (26.3 ± 0.4 g/L) and grape juice (45.6 ± 0.6 g/L); the values agreed well with a conventional F-kit glucose method.