Stability analysis for double‐stranded DNA oligomers and their noncovalent complexes with drugs by laser spray

Abstract

Laser spray, which is a newly developed ionization technique, can characterize the stability of noncovalent complexes in the solution phase. By using this advantage, laser spray has been applied to probe the intrinsic stability of double-stranded DNA (dsDNA) sequences and their binding affinities with various drugs in the solution phase. Systematic experiments were carried out using six 16-mer and three 22-mer dsDNA oligomers, together with the complexes of the 16-mer dsDNA with minor groove binders: berenil, Hoechst 33342, DAPI, and netropsin. Dissociation curves for each dsDNA or each complex were plotted as a function of laser power. The laser power (E50%), where 50% of each dsDNA or each complex was dissociated, was compared with its melting temperature (Tm) determined by UV spectroscopy. Linear correlations between E50% and Tm were obtained not only for the dsDNA oligomers (correlation factor r = 0.9835) but also for the 16-mer dsDNA complexes with minor groove binders (r = 0.9966). In addition, laser spray has successfully clarified the binding affinities of a 16-mer dsDNA with two intercalators: daunomycin and nogalamycin. In the case of the dsDNA-daunomycin complex, by changing the molar ratio of dsDNA : drug from 1 : 1 to 1 : 5, the concentration-dependent stability of the complex was confirmed by laser spray. The present results demonstrate that laser spray mass spectrometry can be a powerful and convenient method to investigate the relative binding affinities of dsDNA-ligand complexes in the solution phase, which could be applied to the early stage of high-throughput screening of drugs targeting for dsDNA.

Thermal unfolding of proteins probed by laser spray mass spectrometry

“…This is consistent with our previous report,25, 28, 31 in which the ion abundances generated by laser spray were found to improve by several orders of magnitude compared with those obtained by conventional ESI. In laser spray, thermal heating is completed instantaneously during the laser irradiation and no fragmentation is observed for the analyzed biomolecules, even when a laser beam with higher laser power (∼2.0 W) irradiates at the tip of the stainless‐steel capillary 30–34. These are the advantages of laser spray in stability analysis of biomolecules in solution.…”

“…These are the advantages of laser spray in stability analysis of biomolecules in solution. Figure 1 indicates that no cleavage of covalent bonds was observed in the laser spray mass spectra of cyt c. Even for thermally labile molecules, such as oligonuleotides, no covalent bond cleavage was observed in laser spray mass spectra 32, 33. Since laser power is not dissipated for the dissociation of covalent bonds, it is possible to simply correlate protein stability with laser power in laser spray mass spectrometry.…”

“…Details of the instrumentation have already been published 25–34. In brief, a sample solution was introduced into a stainless‐steel capillary (i.d.…”

“…One of the advantages of laser spray in the analysis of biomolecules is its convenience; laser spray provides information on the stability of biomolecules instantly just by tuning the laser power. It has been demonstrated that laser power by which 50% of noncovalent complexes are dissociated in a population ( E 50% ) can be an evaluation parameter of binding affinities of protein‐DNA and drug‐DNA complexes in semi‐quantitation experiments by laser spray 32, 33. In the present work, we investigated whether laser spray could be employed to analyze the thermal stability and folding transitions of proteins quantitatively.…”

“…Laser spray, which was developed by Hiraoka and coworkers,25–34 is a hybrid of three basic techniques for the generation of gaseous ions from the condensed phase, i.e., energy‐sudden activation, nebulization and the action of an electric field. Laser spray mass spectrometry can faithfully reflect the solution‐phase characteristics of biomolecules.…”