Dynamic Covalent Programming at DNA Base-Pairing Interfaces

Fluorescent nucleobase analogues (FBAs) are powerful reporters of nucleic acid structure and dynamics, yet strategies for programming their functional behavior directly at the hydrogen-bonding interface of DNA remain limited. Here, we introduce a nucleobase design strategy that enables conformational programming within DNA through site-specific incorporation of a 6-amino adenine analogue (^hzA) using a nosyl-protected phosphoramidite compatible with standard solid-phase synthesis. Postsynthetic condensation with aldehydes generates a structurally diverse library of hydrazone nucleobases with tunable electronic and conformational properties at the N⁶ position. Screening identified exceptionally bright FBAs (up to 4.5 × 10⁴ M^-1 cm^-1), with visible excitation and emission maxima, and quantum yields ranging from 0.004-0.863, depending on conformation, sequence context, local environment, and metal binding. Hydrazone formation can be highly fluorogenic with >120-fold increases in brightness for a coumarin-derived system, enabling real-time monitoring of reaction progress. Kinetic and thermodynamic analyses revealed that product formation and stability are strongly gated by nucleic acid structure: single-stranded DNA (ssDNA) supports rapid formation (k_on ≈ 10 M^-1 s^-1) and highly stable products (K_eq ≈ 10⁷ M^-1), whereas duplex DNA exhibits slower association rates and greater sequence selectivity than ssDNA. Notably, an ortho-phenolic hydrazone (^CySalA) forms an intramolecular hydrogen bond to N⁷ that stabilizes an anti-conformation of the hydrazone, thereby preserving selective pairing with thymidine while gating access to alternative functional states. Correct base pairing suppresses Zn²⁺ coordination and tautomer-dependent spectral transitions of ^CySalA, whereas base pair mismatches restore conformational freedom, enabling metal ion complexation with distinct absorbance/emission signatures. Collectively, this work establishes a general hydrazone-based platform for programming functional states at hydrogen-bonding interfaces, transforming a native hydrogen-bonding face into a modular chemical handle for tuning structure, reactivity, and optical responses within duplex and single-stranded DNA.