Sequence-Dependent Thermodynamic Stability and Frontier-Orbital Reactivity Landscape of the 64 Dna Trinucleotides: A Semi-Empirical Am1 and Conceptual Density Functional Theory Study

The physicochemical behaviour of DNA at the trinucleotide level contributes to sequence-dependent molecular recognition, reactivity, and stability. Here, the complete set of 64 single-stranded DNA trinucleotides (5′-NpNpN-3′) was systematically characterised at the semi-empirical Austin Model 1 (AM1) level using MOPAC2016 (v23.2.5w). Fully optimised geometries were used to determine the heat of formation (ΔHf), dipole moment, frontier molecular orbital (HOMO/LUMO) energies, molecular weight, and COSMO solvent-accessible surface area and volume for every sequence. Koopmans’-theorem relationships applied to the frontier orbital energies were subsequently used to derive conceptual density-functional-theory reactivity descriptors, including ionisation potential, electron affinity, electronegativity, chemical hardness, softness, chemical potential, and global electrophilicity (ω). Across the complete sequence space, ΔHf exhibited a systematic dependence on base identity, with the average thermodynamic ordering A < G < C < T at each nucleotide position. Thymine-rich sequences, exemplified by 5′-TTT-3′ (ΔHf = −1097.5 kcal mol⁻¹), showed the most negative heats of formation, whereas purine-rich sequences such as 5′-AGG-3′ (ΔHf = −660.3 kcal mol⁻¹) showed substantially less negative values. One-way ANOVA indicated a significant effect of the initiating base on both ΔHf (F = 10.62, p < 0.0001) and the HOMO–LUMO gap (F = 4.79, p < 0.005). The HOMO–LUMO gap was inversely related to chemical hardness (r = −1.00), as expected from their mathematical relationship, and showed positive correlations with molecular weight (r = 0.66) and COSMO volume (r = 0.55). Global electrophilicity values covered approximately a 1.6-fold range (ω = 1.85–2.93 eV), with 5′-GTG-3′ exhibiting the highest calculated electrophilicity and 5′-TCA-3′ the lowest. Collectively, these results provide a sequence-resolved computational map of thermodynamic and frontier-orbital-derived reactivity properties across the complete DNA trinucleotide space. The dataset establishes a quantitative AM1/COSMO baseline for investigating how nucleotide composition and sequence context influence the electronic properties of short DNA fragments and provides a foundation for subsequent studies of sequence-dependent chemical reactivity, oxidative processes, and molecular recognition.