Abstract
Understanding the structural conformation and dynamics of nucleic acids in solution is critical for the development of nucleic acid therapeutics involving antisense or mRNA therapies, where controlling stability, efficacy, and manufacturability are important. Circular dichroism (CD) spectroscopy allows rapid and nondestructive solution analysis of the spatial arrangements of nucleic acids. While widely used to analyze protein structure, the potential of CD for DNA- and RNA-based pharmaceutical applications has remained largely unrealized because nucleic acid spectra are complex and difficult to interpret quantitatively. We introduce herein a framework (NA-CDQuant) that transforms CD from a qualitative fingerprinting method into a quantitative tool for nucleic acid structure prediction by combining spectral deconvolution with a rigorously curated synchrotron radiation circular dichroism reference library comprising 18 structurally defined conformational families. This systematic classification overcomes long-standing challenges posed by spectral overlap and structural polymorphism, enabling estimation of the fractional composition of coexisting conformations during real-time monitoring of structural transitions in solution. NA-CDQuant provides mechanistic insight into subtle conformational redistributions that may affect molecular recognition and functional performance assisting pharmaceutical development and medical application by facilitating formulation optimization, stability assessment, quality control, and regulatory support of nucleic acid–based therapeutics.