Research paper Thermodynamics and site stoichiometry of DNA binding by a large antiviral hairpin polyamide Yang Song, Jacquelyn Niederschulte, Kristin N. Bales, James K. Bashkin, Cynthia M. Dupureur * Department of Chemistry & Biochemistry, University of Missouri St. Louis, St. Louis, MO, 63121, USA article info Article history: Received 28 June 2018 Accepted 22 November 2018 Available online 24 November 2018 Keywords: Polyamide DNA Stoichiometry Entropy Antiviral Fluorescence abstract PA1 (dIm-PyPybPyPyPy-g-PyPybPyPyPyPyb-Ta) is a large (14-ring) hairpin polyamide that was designed to recognize the DNA sequence 5’-W 2 GW 7 -3’, where W is either A or T. As is common among the smaller 6-8-ring hairpin polyamides (PAs), it binds its target recognition sequence with low nM affinity. How- ever, in addition to its large size, it is distinct from these more extensively characterized PAs in its high tolerance for mismatches and antiviral properties. In ongoing attempts to understand the basis for these distinctions, we conducted thermodynamics studies of PA1-DNA interactions. The temperature depen- dence of binding affinity was measured using TAMRA-labeled hairpin DNAs containing a single target sequence. PA1 binding to either an ATAT/TATA or an AAAA/TTTT pattern is consistently entropically driven. This is in contrast to the A/T pattern-dependent driving forces for DNA binding by netropsin, distamycin, and smaller hairpin polyamides. Analysis of the salt dependence of PA1-DNA binding reveals that within experimental error, there is no dependence on ionic strength, indicating that the poly- electrolyte effect does not contribute to PA1-DNA binding energetics. This is similar to that observed for smaller PAs. PA1-DNA recognition sequence binding stoichiometries were determined at both nM (fluorescence) and mM (circular dichroism) concentrations. With all sequences and under both condi- tions, multiple PA1 molecules bind the small DNA hairpin that contains only a single recognition sequence. Implications for these observations are discussed. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved. 1. Introduction Hairpin polyamides (PAs) are small polymers that bind the DNA minor groove with high affinity and sequence specificity [1]. Their designs were inspired by the natural products distamycin and netropsin. Over many years, the DNA binding properties of 8-ring PAs have been extensively examined and modified [2e11] with an eye towards their use in the modulation of gene expression [4, 12] and as probes of nucleic acid structure [13]. PAs were designed to capitalize on enthalpically favorable [1] hydrogen bonding with the DNA base pairs in the minor groove. However, due to the properties of bulk water and the peculiarities of DNA as a polyanionic biopolymer, other forces have the potential to significantly contribute to binding interactions. The displace- ment of water molecules can contribute to the favorable entropic driving forces of DNA ligand binding [14]. In addition, electrostatic contacts of protein ligands with the DNA backbone have been structurally characterized and shown in thermodynamic studies to contribute to DNA binding energy [15]. Understanding the relative enthalpic and entropic contributions to ligand binding provides insights into the types of forces that are most important to the strength and specificity of the target inter- action [16, 17]. Through the optimization of favorable interactions and the systematic reduction in unfavorable energetic contribu- tions, the next generation of bioactive PAs can be developed. The binding energetics of minor groove ligands has been recently reviewed [18]. Early thermodynamic studies of polyamide-DNA interactions focused on netropsin and distamycin. In both cases, DNA binding to sequences that feature the more heavily hydrated poly(A)poly(T) pattern has a strong entropic contribution; binding to a sequence that features an alternating A/T pattern is enthalpically driven [19,20]. * Corresponding author. E-mail address: cdup@umsl.edu (C.M. Dupureur). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi https://doi.org/10.1016/j.biochi.2018.11.013 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved. Biochimie 157 (2019) 149e157