To Flip or Not to Flip: Introducing the Universal Flippability Index (UFI) for Epigenetic and Damaged Nucleobases.

The catalogue of chemical modifications on DNA and RNA─from 5-methylcytosine (5mC) and its oxidized derivatives (5hmC, 5fC, 5caC), through oxidative damage lesions (8-oxoguanine, thymine glycol, FapyG, 5-hydroxyuracil), to the RNA epitranscriptomic mark N6-methyladenosine (m6A)─has outpaced our mechanistic understanding of how proteins read, write and erase these marks. Structural biology reveals a striking convergence: DNA glycosylases (UDG, hOGG1, TDG, AAG, MutY, NEIL, NTH1), methyltransferases (DNMT1, M.HhaI), TET dioxygenases and the m6A erasers FTO/ALKBH5 all employ a shared base-flipping strategy. Yet static structural snapshots cannot explain why Thymine DNA Glycosylase rapidly excises 5fC and 5caC from CpG sites while ignoring 5mC and 5hmC, nor why processing rates differ by orders of magnitude between substrates. We propose the Universal Flippability Index (UFI), a computable thermodynamic metric that quantifies the energetic cost of extrahelical base rotation. The UFI decomposes flippability into four physically interpretable components ─ hydrogen-bond disruption (ΔEHB), stacking loss (ΔEStack), steric/backbone-phosphate clash (ΔESteric), and solvent exposure (ΔESolv)─and reports a separate value for each paired conformer, e.g. anti vs syn 8-oxoG or m6A. A high UFI marks a "frustrated" base that breathes out of the helix; a low UFI marks a deeply locked nucleotide. Five case studies illustrate the framework: (i) the TDG-selective epigenetic oxidation gradient (5mC → 5hmC → 5fC → 5caC); (ii) 8-oxoG as a steric "damage beacon"; (iii) NEIL/NTH1 substrates Tg, FapyG and 5-OHU; (iv) etheno-adenine, processed by both AAG (base excision) and ALKBH2 (direct reversal), showing that UFI is necessary but not sufficient; and (v) m6A as a spring-loaded RNA switch. A computational roadmap parametrizes the UFI through quantum-mechanical calculations, molecular-dynamics free-energy simulations, and three orthogonal experimental probes: imino-proton exchange, 19F NMR of 2'-F-labeled substrates, and 31P NMR of the BI↔BII phosphate-backbone equilibrium.