Complexity in the binding of minor groove agents: netropsin has two thermodynamically different DNA binding modes at a single site

Abstract

Structural results with minor groove binding agents, such as netropsin, have provided detailed, atomic level views of DNA molecular recognition. Solution studies, however, indicate that there is complexity in the binding of minor groove agents to a single site. Netropsin, for example, has two DNA binding enthalpies in isothermal titration calorimetry (ITC) experiments that indicate the compound simultaneously forms two thermodynamically different complexes at a single AATT site. Two proposals for the origin of this unusual observation have been developed: (i) two different bound species of netropsin at single binding sites and (ii) a netropsin induced DNA hairpin to duplex transition. To develop a better understanding of DNA recognition complexity, the two proposals have been tested with several DNAs and the methods of mass spectrometry (MS), polyacrylamide gel electrophoresis (PAGE) and nuclear magnetic resonance spectroscopy in addition to ITC. All of the methods with all of the DNAs investigated clearly shows that netropsin forms two different complexes at AATT sites, and that the proposal for an induced hairpin to duplex transition in this system is incorrect.

Microscopic Rearrangement of Bound Minor Groove Binders Detected by NMR

“…Moreover, we can also observe imino peaks that are exchange related (Figure S4, Supplementary Materials). The potential of some minor groove binders to form more than a single 1:1 complex to non-self complementary sequences is further confirmed in a recent study, showing that netropsin forms two individual 1:1 binding modes to an asymmetric DNA hairpin 23 .…”

“…It should be emphasized that these bound state dynamic transitions are in addition to any local thermal fluctuations that occur within a specific binding site. The complexes in this work are for alternating sequence DNA but the literature suggests that different binding site sequences show similar dynamic transitions 23,38–41 . In addition, DB921 and netropsin have very different complexes with the DNA minor groove 26,35,37,39,42–44 and it is also clear that the exchange behavior occurs for many minor groove complex types.…”

“…A recent example is the observation of a “dip” in ITC binding isotherms upon binding of netropsin to a DNA hairpin. Several suggestions including a hairpin to duplex transition were made to explain this unusual behavior and it took a combined approach of mass spectrometry, PAGE gels and NMR to finally reveal that two distinct 1:1 compound-DNA complexes caused the unusual shape of the binding isotherm 23 .…”

“…However, even though a significant amount of binding data is available, the dynamic processes that are involved in minor groove complex formation and maintenance are not fully understood. As part of a recent study that identified more than one compound binding mode at a single DNA minor groove binding site, NMR results were obtained that suggested much faster exchange kinetics between bound compounds than would generally be expected for strong binding groove interactions 23 .…”

Cooperative stabilization of Zn2+:DNA complexes through netropsin binding in the minor groove of FdU-substituted DNA

“…Minor groove binding may impart greater thermodynamic stability to the Zn 2+ :DNA complex and could potentially result in increased uptake into tumor cells through reduced charge density since minor groove binding ligands (Nelson et al, 2007), such as netropsin (Lewis et al 2011), carry positive charges that partially offset the highly negative charge of the phosphodiester backbone of DNA. In this work, we demonstrate that netropsin and Zn 2+ simultaneously occupy the minor and major groove in the DNA hairpin 3’FdU that includes 10 consecutive FdU nucleotides at the 3′-terminus (Fig.…”