Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)] and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities

Loontiens, Frank G.; Regenfuß, Peter; Zechel, Annelies; Dumortier, Lieve; Clegg, Robert M.

doi:10.1021/bi00490a021

Cited by 312 publications

(253 citation statements)

References 34 publications

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“…At 21°C, as well as at 10°C, there is fast exchange of the imino protons of the end base pairs with the solvent molecules (10). This exchange is observed in the 1H NMR spectrum as a broad resonance or, at higher dsDNA concentrations (e.g., 2.5 mM), as a shoulder on a sharp G-C resonance belonging to a nonperipheral base pair (6).…”

Section: Discussionmentioning

confidence: 98%

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Stoichiometry and structure of complexes of DNA oligomers with microgonotropens and distamycin by 1H NMR spectroscopy and molecular modeling.

Blaskó

Bruice

1993

Proc. Natl. Acad. Sci. U.S.A.

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Dien-microgonotropen-c (5c), tren-microgonotropen-b (6b), and distamycin (Dm) bind the A.T-rich region of d(CGCAAATTTGCG)2 (oligo-12) and form 1:1 (5c and 6b) and 2:1 and 4:1 (Dm) complexes. At 1.75 mol ratio of Dm/oligo-12 the 4:1 complex starts to form and coexists with the 2:1 complex and the free double-stranded DNA. No 1:1 and 3:1 complexes were seen, implying a preferential dimeric binding mode of Dm to oligo-12. At 4:1 mol ratio of Dm/oligo-12 there is extensive exchange of the A.T imino protons with the solvent at the binding site. This is presumably due to the opening of the minor groove. Molecular modeling shows that four Dm molecules can fit in a tandem antiparallel way into the minor groove of oligo-12 by widening it to 16-17 A. On going from oligo-12 to the pseudosymmetrical hexadecamer oligo-16 [d(GGCGCAAATTTGGCGG).d(CCGCCAAATTTGCGCC)] the stoichiometry of binding of 5c changes from 1:1 to 2:1. Since oligo-12 and oligo-16 have the same A.T binding site this change in stoichiometry is due to the increase in the G.C terminal pairing. Hoechst 33258 displaces the two 5c molecules bound in the minor groove of oligo-16 at the A.T region. Marked exchange of A.T imino protons was seen in the case of (oligo-16).(Ht)2.

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Section: Discussionmentioning

confidence: 98%

“…Ht (Structure I) is a minor groove binder (9) that becomes highly fluorescent once complexed to dsDNA (10). This property has been used to determine binding constants of different minor groove binders in competition experiments with Ht.…”

Section: Concentrations Of Thementioning

confidence: 99%

Stoichiometry and structure of complexes of DNA oligomers with microgonotropens and distamycin by 1H NMR spectroscopy and molecular modeling.

Blaskó

Bruice

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Immunocytochemistry and DNA measurement The amount of cells per well was measured by a DNA fluorimetric assay based on the binding of Hoechst 33258 dye [17]. At least 6 wells were used per experimental condition, so that triplicate cultures could be used for DNA-extraction and, in parallel, for immunocytochemical analysis of cell composition.…”

Section: Methodsmentioning

confidence: 99%

In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells

Baeyens¹,

Breuck²,

Lardon³

et al. 2004

Diabetologia

289

224

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Aims/hypothesis: Transplantation of insulinproducing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulinproducing cells from adult rat exocrine cells cultured in the presence of growth factors. Methods: Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15. Results: In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulinimmunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft. Conclusions/interpretation: Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

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“…24) Thermodynamic study based on Hoechst 33258-oligonucleotides interactions indicates the positive enthalpy and positive entropy, indicating that binding is driven by hydrophobic interactions with a favorable change in binding free energy. Favorable entropy changes are primarily due to hydrophobic interactions, as a result an increase in solvent entropy from burial of hydrophobic groups and release of water upon binding.…”

mentioning

confidence: 99%

“…12,[22][23][24] Role of water surrounding DNA has emerged as an important entity in the interaction processes. 12,22,25) The network of newly formed hydrogen bonds between the ligand and DNA replaces previously existing direct solute-solvent (water) hydrogen bonds in the minor groove.…”

mentioning

confidence: 99%

Osmolyte Changes the Binding Affinity and Mode of Interaction of Minor Groove Binder Hoechst 33258 with Calf Thymus DNA

Anuradha

Alam

Chaudhury

2010

CHEMICAL & PHARMACEUTICAL BULLETIN

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The bis-benzimidazole molecule Hoechst 33258 ( Fig. 1) is an important DNA minor groove binding ligand.1) Interaction of Hoechst 33258 has been extensively studied using oligonucleotides.2-5) Different aspects of this interaction viz., physicochemical, thermodynamics, NMR, X-ray crystallography, in silico simulation are now well established. 3,[6][7][8][9][10][11] From the classical observation on the ability of Hoechst 33258 binding in the AT rich region of minor groove of DNA. Synthetic chemists have designed various DNA minor groove binders for possible applications such as anticancer, antiviral, and other diseases. 12,13) Studies at cellular level have demonstrated the desired biological effects.14-16) New applications are emerging from studies related to radiosensitization and radioprotection in relation to modification of radiation response by Hoechst derivatives. [17][18][19][20][21] Attempts have also been made to correlate these observations with different Hoechst analogues.Physicochemical and thermodynamic studies have revealed important details on the mechanism of Hoechst 33258-DNA interaction. 12,[22][23][24] Role of water surrounding DNA has emerged as an important entity in the interaction processes. 12,22,25) The network of newly formed hydrogen bonds between the ligand and DNA replaces previously existing direct solute-solvent (water) hydrogen bonds in the minor groove. Hydration changes strongly influences the thermodynamics of ligand-DNA interaction. The change in hydration after the complex formation appeared to be governed by the structure of the ligand. 3,[26][27][28][29] Most of the studies and hence our understandings are still based on oligonucleotides. 5,27,30) Water molecules located close to the binding surfaces are more structured than those of bulk solvent. These locally structured water molecules are retained or released during a binding process with different thermodynamic consequences. Various techniques are available to study hydration changes accompanying ligand-DNA interaction 31,32) and osmotic stress approach has gained importance due to its ability to specifically alter water activity of the surrounding solution; a fact that has been harnessed to measure forces between molecules. 31,32) It is relatively a simple technique to study the hydration changes upon ligand-DNA interaction and gives stoichiometric amount of water release or uptake upon biomolecular complexation.27) The principle of osmotic stress method is based on Gibbs-Duhem equation. 34,35) The neutral solutes are added directly to the solution containing the ligand and macromolecule causes changes in the water activity of the solution. Osmotic stress method has been employed for studying the hydration changes for minor groove binder as well as for intercalators. 5,26,27,[36][37][38] In living system, small molecules interact with DNA in a very crowded environment. The presence of co-solutes in the in-vivo conditions affects the interaction process therefore it is important to study the interaction of Hoechst 33258...

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Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)] and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities

Cited by 312 publications

References 34 publications

Stoichiometry and structure of complexes of DNA oligomers with microgonotropens and distamycin by 1H NMR spectroscopy and molecular modeling.

Stoichiometry and structure of complexes of DNA oligomers with microgonotropens and distamycin by 1H NMR spectroscopy and molecular modeling.

In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells

Osmolyte Changes the Binding Affinity and Mode of Interaction of Minor Groove Binder Hoechst 33258 with Calf Thymus DNA

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