Role of Water in Netropsin Binding to an A₂T₂ Hairpin DNA Site: Osmotic Stress Experiments

Abstract

The formation of two different minor groove complexes between netropsin and A2T2 DNA has been attributed to specific binding and hydration effects. In this study, we have examined the effect of added osmolyte (e.g., TEG or betaine) on the binding of netropsin to a hairpin DNA, d(CGCGAATTCGCGTC-TCCGCGAATTCGCG)-3, having a single A2T2 binding site. Netropsin binding to this DNA construct is described by a two fractional site model with a saturation stoichiometry of 1:1. Free energy changes, ΔGi, for formation of both complex I and complex II decrease continuously as osmolyte is added (e.g., ΔG1 decreases by 1.3 kcal/mol and ΔG2 decreases by 0.8 kcal/mol in 4 m osmolyte vs buffer). The negative ΔCp values for formation of both complexes, I and II, are largely unaffected by the addition of osmolyte. Formation of complex I is accompanied by the acquisition of 31 water molecules vs 19 waters for complex II. The most significant difference between the two osmolytes is that betaine diminishes the fractional formation of the complex II species, virtually eliminating complex II at 2 m. Addition of osmolyte or a decrease in the temperature have approximately the same effect on DNA hydration and on the thermodynamics of netropsin binding.

Mechanism for the Binding of Netropsin to Hairpin DNA Revealed Using Nanoscale Ion Emitters in Native Mass Spectrometry

“…Netropsin binds deep in the minor groove of DNA primarily to AT-rich sequences of double stranded DNA by hydrogen bonding, van der Waals, and electrostatic interactions based on X-ray crystallographic structures (Supporting Information (SI) Figure S1). ,, Netropsin binds to DNA particularly favorably at duplexed AATT sites compared to other AT-rich sequences that have been investigated. − , However, the mechanism of netroposin binding to hairpin DNA in solution remains controversial, and two competing mechanism have been actively debated in the literature. , In one mechanism, netropsin can bind in two different modes to a single duplexed AATT binding site of hairpin DNA; that is, the two-mode (2M) mechanism (Figure a) . Although this mechanism is supported by results from high-resolution isothermal titration calorimetry (ITC), , native polyacrylamide gel electrophoresis (PAGE), − , and conventional electrospray ionization mass spectrometry (ESI-MS), the two specific binding modes have not been directly detected.…”

“…Small molecules that bind to the minor groove of DNA are of interest in the development of biomedical applications, including the regulation of gene expression and the discovery and advancement of potential anticancer, antibiotic, and antiviral therapies. − One of the first small molecules that was identified to bind selectively to the minor groove of specific DNA sequences is netropsin, which is a naturally occurring γ-peptide that was first isolated from Streptomyces netropsis in 1951 . Netropsin is the basic scaffold for many analogues that have been synthesized, including combinatorially, to investigate and modulate ligand–DNA interactions. ,− An understanding of the mechanism of interactions between ligands and DNA is of fundamental importance in DNA-based rational drug design and in molecular recognition. ,, …”

“…Here, nanoscale ion emitters in native mass spectrometry were used to significantly reduce the adduction of salt to ligand–DNA oligonucleotide complex ions, including in the presence of relatively high concentrations of nonvolatile salts that can stabilize some DNA–ligand interactions in solution. Two different hairpin oligonucleotides that each have a netropsin binding site (duplexed AATT) were chosen because these oligonucleotides have been used previously to investigate the mechanism of netropsin binding to the minor groove of DNA. − , By increasing the mass spectral sensitivity owing to reduced salt adduction using nanoelectrospray ion emitters with inner tip diameters of ∼250 nm, the simultaneous binding of two netropsin molecules to both the hairpin and duplex forms of H1 (5′-CGA­ATTCC­TCTCG­GAAT­TCG-3′-6068 Da) and H2 (5′-CGCA­ATTG­GCTCTC­GCCAAT­TGCG-3′-7304 Da) can be readily observed; that is, the native mass spectrometry data obtained here indicate that netropsin binds to DNA by a different mechanism than the 2M and HD mechanisms under these conditions, including at initial concentrations of DNA and netropsin that are comparable to (10 μM; 10 pmol/μL) and significantly lower (500 nM; 0.5 pmol/μL) than that used in previous studies. − , In addition, the sequential and simultaneous binding of netropsin to both hairpin and duplex DNA oligonucleotides that were extracted from both the duplex and hairpin DNA bands in native PAGE gels that contain high concentrations of nonvolatile surfactants and salts was observed using native MS. Based on results from both native MS and PAGE, the presence of netropsin does not significantly affect the relative abundances of hairpin DNA compared to that for the duplex of DNA. Using analytically derived equations and nanoscale ion emitters, the binding affinities for five different equilibria in the simultaneous and sequential binding of netropsin to the hairpin and duplex forms of H1 and H2 can be obtained by directly measuring the corresponding netropsin–DNA oligonucleotide complexes in native MS.…”

“…6,13,14 Netropsin binds to DNA particularly favorably at duplexed AATT sites compared to other ATrich sequences that have been investigated. [7][8][9][10]15 However, the mechanism of netroposin binding to hairpin DNA in solution remains controversial, and two competing mechanism have been actively debated in the literature. 7,9 In one mechanism, netropsin can bind in two different modes to a single duplexed AATT binding site of hairpin DNA; that is, the two-mode (2M) mechanism (Figure 1a).…”

“…7,9 In one mechanism, netropsin can bind in two different modes to a single duplexed AATT binding site of hairpin DNA; that is, the two-mode (2M) mechanism (Figure 1a). 7 Although this mechanism is supported by results from high-resolution isothermal titration calorimetry (ITC), 7,9 native polyacrylamide gel electrophoresis (PAGE), [7][8][9][10]15 and conventional electrospray ionization mass spectrometry (ESI-MS), 7 the two specific binding modes have not been directly detected. In an alternative mechanism, the binding of a ligand to hairpin DNA containing a duplexed AATT binding site can induce a transition from hairpin DNA to duplex DNA with an internal "bulge" in place of the hairpin loop; that is, a ligand-binding induced hairpin-to-duplex (HD) transition mechanism (HD Mechanism; Figure 1b).…”