Wavelet-domain multiway spectral separation of free drug, DNA, and drug–DNA complex profiles for quantitative binding analysis based on fractional occupancy ( <i>θ</i> )

Classical UV spectrophotometric analysis of drug–DNA interactions is fundamentally limited by severe spectral overlap and by linearization schemes relying on molar absorptivity assumptions. Under such conditions, conventional approaches, including the Benesi–Hildebrand method, often yield ambiguous or underestimated binding parameters. In this work, the UV spectroscopic binding analysis is reformulated within a wavelet-assisted multiway framework that enables resolution of highly overlapped UV spectra of the free drug, DNA, and the drug-DNA complex. Binding analysis is performed using the fractional occupancy ratio θ/(1 − θ), providing an assumption-free description of the drug-DNA equilibrium. UV absorption spectra recorded during the titration of hydroxychloroquine (HYD) with DNA were transformed into the wavelet domain using the continuous wavelet transform (CWT) and arranged as a three-way tensor. Parallel factor analysis (PARAFAC) decomposition enabled unique resolution of free HYD, free DNA, and the HYD-DNA complex without reference spectra or additional spectroscopic assumptions. Fractional occupancy values increased from 0.39 to 0.58 as DNA concentration increased from 4.0 × 10−5 to 2.0 × 10−4 M, indicating a consistent binding process. Binding constants evaluated using three definitions of free DNA ranged from (9.6 ± 3.8) × 10³ to (2.28 ± 1.42) × 104 M−1. In contrast, classical Benesi–Hildebrand analysis yielded a substantially lower apparent binding constant (∼1.77 × 10³ M−1), underscoring the limitations of single-wavelength linearization in overlapped systems. The proposed wavelet-PARAFAC framework provides a robust, assumption-free strategy for resolving highly overlapped UV spectra and for quantitative drug–DNA binding analysis directly from UV spectroscopic data.