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Nucleic acids can adopt multiple higher-order structures (HOSs) from a single sequence, and such structural polymorphism may change in a concentration-dependent manner. This suggests that nucleic acid therapeutics may undergo concentration-dependent structural changes under formulation-relevant conditions. However, systematic evaluation of this behavior has been limited because spectroscopic techniques are typically applied over different concentration ranges. Here, we propose an orthogonal spectroscopic assessment strategy that enables CD, Raman, and FT-IR spectroscopy to be performed under closely matched concentration conditions. Using the thrombin-binding aptamers, we investigated concentration-dependent changes in HOS over a formulation-relevant concentration range. CD combined with reference-based principal component analysis revealed distinct concentration-dependent structural transitions. Complementary Raman and FT-IR analyses provided insights into nucleobase orientation, hydrogen-bonding environments, and phosphate backbone conformations, supporting the CD-based interpretations. Notably, one sequence showed G4-like spectroscopic features associated with higher-order assembly at high concentrations, despite its low predicted G4-forming propensity based on sequence information alone. These results indicate that concentration-dependent structural polymorphism should be evaluated in the characterization of nucleic acid therapeutics. The strategy presented here provides a practical platform for evaluating HOS under formulation-relevant conditions and supports the development and quality assessment of nucleic acid therapeutics.

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• Creative Commons BY Attribution 4.0 International
  

Keyword: Circular Dichroism spectrum, Raman spectrum, Fourier Transform Infrared spectrum, Fluorescence spctrum, Atomic Force Microscope

Date published:

Publisher: National Institute for Materials Science

Journal:

• Molecular Therapy Nucleic Acids (ISSN: 21622531)

Funding:

Manuscript type: Not a journal article

MDR DOI: https://doi.org/10.48505/nims.6486

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Updated at: 2026-08-26 13:26:50 +0900

Published on MDR: 2026-08-26 14:28:09 +0900