Evaluation of binding of perylene diimide and benzannulated perylene diimide ligands to dna by electrospray ionization mass spectrometry

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The DNA binding of novel threading bis-intercalators V1, trans-D1, and cis-C1, which contain two naphthalene diimide (NDI) intercalation units connected by a scaffold, was evaluated using electrospray ionization mass spectrometry (ESI-MS) and DNAse footprinting techniques. ESI-MS experiments confirmed that V1, the ligand containing the -Gly 3 -Lys-peptide scaffold, binds to a DNA duplex containing the 5'-GGTACC-3' specific binding site identified in previous NMR-based studies. The ligand formed complexes with a ligand/DNA binding stoichiometry of 1:1, even when there was excess ligand in solution. Trans-D1 and cis-C1 are new ligands containing a rigid spirotricyclic scaffold in the trans-and cis-orientations, respectively. Preliminary DNAse footprinting experiments identified possible specific binding sites of 5'-CAGTGA-5' for trans-D1 and 5'-GGTACC-3' for cis-C1. ESI-MS experiments revealed that both ligands bound to DNA duplexes containing the respective specific binding sequences, with cis-C1 exhibiting the most extensive binding based on a higher fraction of bound DNA value. Cis-C1 formed complexes with a dominant 1:1 binding stoichiometry, whereas trans-D1 was able to form 2:1 complexes at ligand/DNA molar ratios ≥ 1 which is suggestive of non-specific binding. Collisional activated dissociation (CAD) experiments indicate that DNA complexes containing V1, trans-D1, and cis-C1 have a unique fragmentation pathway, which was also observed for complexes containing the commercially available bisintercalator echinomycin, as a result of similar binding interactions, marked by intercalation in addition to hydrogen bonding by the scaffold with the DNA major or minor groove.

Section: Complexes With Trans-d1 and Cis-c1mentioning

confidence: 99%

Screening of threading bis-intercalators binding to duplex DNA by electrospray ionization tandem mass spectrometry

Mazzitelli

Chu

Reczek

et al. 2007

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“…During the ESI process, noncovalent complexes in solution are transferred to the gas phase with minimal internal energy, allowing many of the binding interactions to be maintained. The preservation of these noncovalent complexes allows information about binding stoichiometry and selectivity to be elucidated from the mass spectra Our group [16,17] and others [18 -24] have focused on developing ESI-MS based techniques to assess whether the binding of quadruplex interactive ligands observed in the gas-phase can be correlated to known solution behavior with the ultimate goal of developing ESI-MS as a screening tool for drug/DNA complexes.…”

mentioning

confidence: 99%

Gas-phase stability of G-quadruplex DNA determined by electrospray ionization tandem mass spectrometry and molecular dynamics simulations

Mazzitelli

Wang²,

Smith

et al. 2007

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) 4 , were investigated using molecular dynamics simulations and electrospray ionization mass spectrometry (ESI-MS). MD simulations revealed that the G-quadruplexes maintained their structures in the gas phase although the G-quartets were distorted to some degree and ammonium ions, retained by [d(TG 4 T)] 4 and [d(T 2 G 3 T)] 4 , played a key role in stabilizing the tetrad structure. Energyvariable collisional activated dissociation was used to assess the relative stabilities of each quadruplex based on E 1/2 values, and the resulting order of relative stabilities was found to beThe stabilities from the E 1/2 values generally paralleled the RMSD and relative free energies of the quadruplexes based on the MD energy analysis. One exception to the general agreement is [d(G 4 T 4 G 4 )] 2 , which had the lowest E 1/2 value, but was determined to be the most stable quadruplex according to the free-energy analysis and ranked fourth based on the RMSD comparison. This discrepancy is attributed to differences in the fragmentation pathway of the quadruplex. T he basis of many anticancer and antitumor therapies is the interaction between small molecule drugs and nucleic acid structures. While most current DNA-interactive therapies target duplex DNA, G-quadruplex DNA has attracted recent interest as a potential anti-cancer drug target because of its role in telomere maintenance [1][2][3]. Telomeres are the regions of non-coding DNA found on the extremities of chromosomes [4]. In addition to protecting the chromosomes from fusion and degradation, telomeres allow for the complete replication of the chromosomal DNA. With each subsequent cell division process, the length of the telomeres is shortened until a critical length is reached, leading to cell senescence and death [5]. Telomerase is a reverse transcriptase enzyme that is responsible for maintaining the length of telomeric DNA. While this enzyme is inactive in most human somatic cells, high levels of telomerase activity are found in 80% to 90% of human cancer cells [6,7].Telomeric DNA is composed of tandem repeats of G-rich sequences, such as the d(T 2 AG 3 ) n sequence in mammals [8], d(T 2 G 4 ) n in Tetrahymena [9], and d(T 4 G 4 ) n in Oxytricha [10]. These and other G-rich sequences have been shown to form G-quadruplex structures in vitro. G-quadruplex DNA forms via Hoogsteen hydrogen bonding between a planar arrangement of four guanine nucleobases, termed a G-quartet. In the quadruplex structure, the G-quartets stack on top of one another and the overall structure is stabilized by monovalent cations such as Na ϩ , K ϩ , or NH 4 ϩ coordinated in the central cavity of the tetrad [11]. Structural polymorphism resulting from different quadruplex sequences and strand orientations has been demonstrated in vitro [12]. A DNA sequence containing a single G-rich repeat can form a four-stranded parallel G-quadruplex. Strands containing two or more G-rich regions can form G-G hairpins and dimerize in different orientations to form a two-stranded quadruplex, while a sequence w...

“…In contrast, gas-phase activation of small ligand-nucleic acid complexes has been more actively pursued to investigate their stability rather than to map the actual binding sites [52][53][54][55][56]. Characteristic dissociation patterns obtained from these precursor ions have been effectively correlated to the nature and selectivity of the binding interaction.…”

mentioning

confidence: 99%

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry

Turner

Hagan

Kohlway

et al. 2006

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (⌿-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the ⌿-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC·SL3 and NC·SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC·SL2 complex. . The relatively low mutation frequency of this ϳ120 nt stretch of genomic RNA makes the packaging signal a very promising target for the development of new therapeutic strategies to contend with the rapid emergence of drug resistant strains [7,8]. For this reason, steps have been made toward the identification of possible inhibitors that may bind specific ⌿-RNA structures and disrupt their normal functions [9 -11]. In a systematic investigation of classic nucleic acid ligands, we have recently shown that aminoglycosidic antibiotics are not only capable of binding individual domains of ⌿-RNA, but can also inhibit their interactions with NC in a structure-specific fashion [12]. Initial tests to locate the actual binding sites on the different RNA substrates, which is crucial to explain the mechanism of preferential inhibition, were based on the analysis of mutant-ligand complexes performed by electrospray ionization (ESI) [13,14] and Fourier transform mass spectrometry (FTMS) [15,16].The favorable energetics characteristic of ESI enable the analysis of relatively labile noncovalent complex...