Second Harmonic Scattering Signature of Adenosine-Thymidine Pairing in Water

Deoxyribonucleic acid (DNA) is the key core molecule responsible that stores, copies and transmits the genetic information in living organisms. It ensures heredity, cellular function and evolutionary adaptation. DNA double-helix stability is determinant to preserve its function avoiding mutations. Moreover, understanding the molecular interactions governing adenosine–thymidine (AT) base pairing is essential for elucidating the mechanisms underlying the stability and structural organization of DNA. Thus, in this work, we demonstrate that second harmonic scattering (SHS), also known as hyper-Rayleigh scattering, provides a highly sensitive approach for probing adenine–thymine (A–T) base pairing in aqueous solution at room temperature. SHS intensity and polarization-resolved measurements were performed on aqueous neat solutions of deoxyadenosine monophosphate (dAMP) and thymidine monophosphate (dTMP) during controlled mixing experiments. Progressive changes in the nonlinear optical response, including a decrease in SHS intensity and an evolution of the depolarization ratio, reveal the formation of A–T complexes under ambient conditions. A complexation model was developed to quantify the fraction of duplex and extract the nonlinear optical response of A-T. The experimental results indicate an effective pairing free energy of approximately −3 kcal mol–1, consistent with the weak and transient nature of isolated nucleotide association in water. Density functional theory calculations performed at the B3LYP/6–31G(d) level with implicit solvent support the experimental observations by reproducing the hyperpolarizability, depolarization ratio of the A–T duplex. These results establish SHS as a powerful, noninvasive technique for detecting weak biomolecular interactions in solution and provide new opportunities for investigating nucleotide base pairs recognition and supramolecular assembly under physiologically relevant conditions. Altogether, our study demonstrates that SHS can quantify AT pairing with accuracy, revealing about their interaction. It establishes SHS as a highly sensitive, noninvasive technique for probing weak biomolecular interactions under physiologically relevant conditions.