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Updated: Jun 28, 2026

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Published on: November 22, 2014
Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: a case study with
Burki Rajendar1, Arivazhagan Rajendran, Yusuke Sato
1Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.
Abstract:
Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 x 10(6) M(-1) and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 x 10(6) M(-1) and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand-nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand-nucleotide interaction, which leads to the improved binding affinity.
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