A novel approach using electrospray ionization mass spectrometry to study competitive binding of small molecules with mixed DNA sequences

Abstract

Minor groove binding compounds have been shown to induce changes in global DNA conformation, allosterically inhibiting DNA-protein interactions necessary for transcriptional processes. Many minor groove binders are specific for AT-base pairs but have little preference over alternating AT or A-tract sequences. Few compounds, other than polyamides, show selectivity for mixed sequences with AT and GC base pairs. Electrospray ionization mass spectrometry (ESI-MS) can provide insight on the stoichiometry and relative affinities in minor groove recognition of different DNA sequences with a library of minor groove binders. A goal in our current research is to develop new compounds that recognize mixed sequences of DNA. In an effort to optimize screening for compounds that target mixed AT and GC base pair sequences of DNA, ESI-MS was used to study the competitive binding of compounds with a mixed set of DNA sequences. The method identified preferred binding sites, relative affinities, and concentration-dependent binding stoichiometry for the minor groove binding compounds netropsin and DB75 with AT-rich sequences, and DB293 with ATGA and AT-sites.

Mixed up minor groove binders: Convincing A·T specific compounds to recognize a G·C base pair

“…41,42 The use of multiple DNA sequences simultaneously mixed with a small molecule creates a competitive binding environment for comparison of DNA-ligand interactions. Since the free ligand concentration for all of the DNAs is the same, peak intensities of the free, unbound DNA and the DNA-small molecule complexes offer insight into the binding mode and preferred binding site(s).…”

Resolution of Mixed Site DNA Complexes with Dimer‐Forming Minor‐Groove Binders by Using Electrospray Ionization Mass Spectrometry: Compound Structure and DNA Sequence Effects

“…However, neither of these two systems is preferred over the furan found in 1 based on results obtained using ESI-MS with mixed sequences. [10] …”

“…The peak intensities and Δ T m values for 1 and its analogs with initial mixed sequences are compared in Figure 10A with a superimposed model of the compounds (Figure 10B) to illustrate differences in structural conformations. The values for 1 and ATGA were taken from previously published results using ESI-MS. [10] A comparison of Δ T m values for 1 with ATGA (Table S2) shows that 1 prefers TTGA and ATGA over ATGT and AGTA. This is consistent with the results obtained using ESI-MS, as shown in Figure 9, which indicates that the choice and arrangement of base pairs in the target site plays a key role in forming complexes between the parent compound 1 and DNA.…”

“…The level of cooperativity is still observed, and is in agreement with earlier reports from our group demonstrating the cooperative binding of 1 to ATGA by ESI-MS using 5 μ m of DNA. [10] For our systems, there is a general preference for using 5 μ m of DNA since it results in a larger signal for the DNA and/or complexes over using 2.5 μ m . A spectrum using 2.5 μ m concentrations of DNA with compound 1 can be found in the Electronic Supporting Information (Figure S5).…”

“…We recently reported a high-throughput method using ESI-MS to simultaneously screen multiple DNA-minor groove binder interactions. [10] This technique is advantageous over other screening methods because ESI-MS is gentle enough to detect complexes yet powerful enough to sort out similar complexes. The complexes detected are of minor groove binding compounds having relatively high binding affinities so that they can be detected at low concentrations.…”