Recognition Mechanism of Complementary Nucleobases and Sequences in DNA and RNA: Interplay of Watson-Crick Hydrogen Bond Formation and Base Stacking Interactions

Review article, 2026

A/T(U) and G/C nucleobase pair formation in DNA and RNA is crucial to numerous fundamental biological processes, including replication, transcription, and translation. The specificity of A/T(U) and G/C base pairing is used for the recognition of complementary sequences in medical and biotechnological applications, such as PCR, nucleic acid drugs, and CRISPR-Cas9-based gene editing. It is essential to understand and predict fidelity of biological reactions, avoiding off-target binding, in order to improve the accuracy and efficacy of applications. In particular, recognition mechanisms of complementary bases or whole sequences must be understood in detail. Despite the prevailing view that Watson-Crick hydrogen bonding is a primary mechanism for complementary base recognition, several experiments have shown that DNA polymerase does not require hydrogen bonding to select complementary bases. Other factors, such as the shape and geometric fitting of the bases and the base stacking, also appear to be crucially involved in the selection. E.g., artificial bases lacking the ability to form hydrogen bonds can still be recognized by DNA polymerase solely based on base-pair geometry. However, hydrogen bonding also contributes importantly to recognition. The accuracy of selecting a complementary nucleobase or sequence varies depending on reactions, suggesting the co-existence of multiple selection mechanisms. This review provides an overview of biological processes and applications involving base pairing and discusses the molecular mechanism underlying complementary base recognition.