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1.
Biopolymers ; 99(12): 1006-18, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23536479

ABSTRACT

The 3'-terminal extensions of eukaryotic chromosomes are unique examples of functional single-stranded DNA. Human telomeres are constructed of the repeated DNA sequence 5'-d(TTAGGG). Four-repeats of human telomeric DNA have been characterized by high-resolution techniques to be capable of forming at least five distinct monomeric conformations. The predominant solution topology is influenced by solution conditions and the presence of 3'- or 5'-flanking residues. This study describes the unfolding mechanisms for human telomeric quadruplexes formed by eight sequence variants that form three unique antiparallel topologies in K(+) solution. Thermal unfolding monitored by circular dichroism is analyzed by singular value decomposition to enumerate the number of significant spectral species required to model the unfolding process. Thermal denaturation of all quadruplexes studied is found to be best modeled by a four-state sequential mechanism with two populated intermediates. The thermal unfolding was also investigated in 50% (v/v) acetonitrile in which a parallel topology is favored. Under these dehydrating conditions, quadruplex thermal denaturation is best modeled by a three-state sequential unfolding mechanism with one populated intermediate. Dehydrated parallel quadruplexes demonstrate increased thermal stability. The spectral properties of the unfolding intermediate suggest that it is most likely a triple-helical structure.


Subject(s)
G-Quadruplexes , Telomere , Circular Dichroism , DNA/chemistry , DNA, Single-Stranded , Humans , Nucleic Acid Conformation , Thermodynamics
2.
Biochimie ; 93(8): v-vi, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21782066
3.
Biochimie ; 93(8): 1297-309, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21679743

ABSTRACT

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)(2) forms parallel bimolecular GQ in the presence of ≥66 mM K(+); GQs are not formed under Na(+), Li(+) or low K(+) conditions. The thermodynamic parameters for GQ folding at 60 µM oligonucleotide and 100 mM KCl are ΔH = -35 ± 2 kcal mol(-1) and ΔG(310) = -1.4 kcal mol(-1). Quadruplex [d(TAGGG)(2)](2) binds 2-3 K(+) ions with K(d) of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)(2) from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)(2), which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ~10(6) M(-1) binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG(298) of -8.0 kcal mol(-1). TMPyP4 binds with 3:1 stoichiometry and K(a) of ~10(6) M(-1). ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)(2)](2) displaying ΔT(1/2) of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT(1/2) = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT(1/2) = -2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.


Subject(s)
G-Quadruplexes , Metalloporphyrins/chemistry , Zinc/chemistry , Zinc/metabolism , Binding Sites , Circular Dichroism , Fluorescence Resonance Energy Transfer , Freezing , Lithium/chemistry , Lithium/metabolism , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Potassium/chemistry , Potassium/metabolism , Sodium/chemistry , Sodium/metabolism , Thermodynamics
4.
Nucleic Acids Res ; 36(17): 5482-515, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18718931

ABSTRACT

In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.


Subject(s)
G-Quadruplexes , Ions/chemistry , Kinetics , Models, Molecular , Solvents/chemistry , Thermodynamics
5.
Biophys Chem ; 126(1-3): 186-96, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16837123

ABSTRACT

Isothermal titration calorimetry, ITC, has been used to determine the thermodynamics (DeltaG, DeltaH, and -TDeltaS) for binding netropsin to a number of DNA constructs. The DNA constructs included: six different 20-22mer hairpin forming sequences and an 8-mer DNA forming a duplex dimer. All DNA constructs had a single -AT-rich netropsin binding with one of the following sequences, (A(2)T(2))(2), (ATAT)(2), or (AAAA/TTTT). Binding energetics are less dependent on site sequence than on changes in the neighboring single stranded DNA (hairpin loop size and tail length). All of the 1:1 complexes exhibit an enthalpy change that is dependent on the fractional saturation of the binding site. Later binding ligands interact with a significantly more favorable enthalpy change (partial differential DeltaH(1-2) from 2 to 6 kcal/mol) and a significantly less favorable entropy change (partial differential (-TDeltaS(1-2))) from -4 to -9 kcal/mol). The ITC data could only be fit within expected experimental error by use of a thermodynamic model that includes two independent binding processes with a combined stoichiometry of 1 mol of ligand per 1 mol of oligonucleotide. Based on the biophysical evidence reported here, including theoretical calculations for the energetics of "trapping" or structuring of a single water molecule and molecular docking computations, it is proposed that there are two modes by which flexible ligands can bind in the minor groove of duplex DNA. The higher affinity binding mode is for netropsin to lay along the floor of the minor groove in a bent conformation and exclude all water from the groove. The slightly weaker binding mode is for the netropsin molecule to have a slightly more linear conformation and for the required curvature to be the result of a water molecule that bridges between the floor of the minor groove and two of the amidino nitrogens located at one end of the bound netropsin molecule.


Subject(s)
DNA/chemistry , Netropsin/chemistry , Nucleic Acid Conformation , Thermodynamics , Base Sequence , Calorimetry , Ligands , Oligonucleotides/chemistry , Protein Conformation
6.
Methods Enzymol ; 340: 3-22, 2001.
Article in English | MEDLINE | ID: mdl-11494856

ABSTRACT

Binding studies provide information of fundamental and central importance for the complete understanding of ligand-DNA interactions. Studies of ligand binding to long natural DNA samples, to synthetic deoxypolynucleotides of simple repeating sequence, and to oligonucleotides of defined sequence are all needed to begin to understand the interaction in detail. Binding studies provide entry into the thermodynamics of the DNA interactions, which in turn provides great insight into the molecular forces that drive the binding process. This chapter summarizes both model-dependent and -independent approaches for the analysis and interpretation of binding isotherms, and should serve as a concise guide for handling experimental data.


Subject(s)
DNA/metabolism , Animals , Base Sequence , Biophysical Phenomena , Biophysics , Cattle , DNA/chemistry , DNA Adducts/chemistry , DNA Adducts/metabolism , Daunorubicin/chemistry , Daunorubicin/metabolism , In Vitro Techniques , Kinetics , Ligands , Models, Biological , Thermodynamics
10.
Bioorg Med Chem ; 9(5): 1141-8, 2001 May.
Article in English | MEDLINE | ID: mdl-11377172

ABSTRACT

Herein we report the synthesis and characterization of a polyintercalator with eight potential intercalating l,4,5,8-naphthalenetetracarboxylic diimide (NDI) units linked in a head-to-tail arrangement via a peptide linker. UV spectroscopy and viscometry measurements indicated the molecule binds to double-stranded DNA with all eight NDI units intercalated simultaneously. Competition dialysis and DNAse 1 footprinting studies revealed a preference for GC-rich regions of DNA, and circular dichroism studies revealed significant distortion of B-form DNA upon binding. Our so-called "octamer" represents, to the best of our knowledge, the first intercalator that binds as an octakis-intercalator, capable of spanning at least 16 base pairs of DNA.


Subject(s)
DNA/chemistry , GC Rich Sequence/physiology , Imides/chemistry , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Naphthalenes/chemistry , Animals , Base Pairing/genetics , Base Pairing/physiology , Binding Sites/physiology , Cattle , Circular Dichroism , DNA/metabolism , DNA Footprinting/methods , GC Rich Sequence/genetics , Imides/metabolism , Intercalating Agents/chemical synthesis , Naphthalenes/metabolism , Peptides/chemistry , Spectrophotometry, Ultraviolet/methods , Viscosity
11.
J Am Chem Soc ; 123(1): 1-7, 2001 Jan 10.
Article in English | MEDLINE | ID: mdl-11273594

ABSTRACT

The hydration changes that accompany the DNA binding of five intercalators (ethidium, propidium, proflavine, daunomycin, and 7-aminoactinomycin D) were measured by the osmotic stress method with use of the osmolytes betaine, sucrose, and triethylene glycol. Water uptake was found to accompany complex formation for all intercalators except ethidium. The difference in the number of bound water molecules between the complex and the free reactants (Deltan(w)) was different for each intercalator. The values found for Deltan(w) were the following: propidium, +6; daunomycin, +18; proflavine, +30; and 7-aminoactinomycin D, +32. For ethidium binding to DNA a value of Deltan(w) = +0.25(+/-0.3) was found, indicating that within experimental error no water was released or taken up upon complex formation. Intercalation association constants measured in D2O were found to increase relative to values measured in H2O for all compounds except ethidium. A positive correlation between the ratio of binding constants (K(D2O)/K(H2O)) and Deltan(w) was found. These combined studies identify water as an important thermodynamic participant in the formation of certain intercalation complexes.


Subject(s)
DNA/chemistry , Intercalating Agents/chemistry , Water/chemistry , Osmosis/drug effects
12.
J Biol Chem ; 276(6): 4028-37, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11056158

ABSTRACT

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.


Subject(s)
Bombyx/enzymology , Glycine-tRNA Ligase/chemistry , Animals , Biopolymers , Circular Dichroism , Protein Denaturation , Spectrometry, Fluorescence , Tryptophan/chemistry
13.
Anticancer Drug Des ; 16(2-3): 99-107, 2001.
Article in English | MEDLINE | ID: mdl-11962518

ABSTRACT

Indolocarbazoles derived from the antibiotic rebeccamycin represent an important group of antitumor agents. Several indolocarbazoles are currently undergoing clinical trials. These compounds inhibit topoisomerase 1 to produce DNA breaks that are responsible for cell death. Unlike classical topoisomerase I poisons like camptothecin, glycosyl indolocarbazoles can form stable complexes with DNA even in the absence of topoisomerase I. At least in part, their mode of action is reminiscent of that of the anthracyclines, which also bind to nucleic acids and interfere with topoisomerase II. The lead synthetic compound in the series is the uncharged drug NB-506, which bears a glucose residue attached to the indolocarbazole chromophore substituted with two hydroxyl groups at positions 1 and 11. Here we report a detailed biophysical study aimed at characterizing the DNA binding properties of NB-506. Molecular modeling was used to compare the conformation and electronic properties of NB-506 and its analogue ED-571 bearing the two hydroxyl groups at positions 2 and 10. Surface plasmon resonance experiments, performed with DNA hairpin oligomers, indicate that NB-506 binds almost equally well to both AT and GC base pairs, and the binding affinity (K = 10(5) M(-1)) is similar to that of certain classical intercalators such as amsacrine and bisantrene. Isothermal titration calorimetry experiments show that the binding of NB-506 is enthalpy-driven (deltaH = -7.2 kcal/mol). The binding enthalpy measured for NB-506 is similar to that obtained with doxorubicin but the DNA interaction processes for the two drugs differ markedly in terms of entropy and deltaG. The free energy of NB-506 binding to DNA is considerably less favorable than that of doxorubicin. These biophysical data help us to understand further how rebeccamycin-type anticancer drugs interact with DNA.


Subject(s)
Aminoglycosides , Antineoplastic Agents/pharmacology , Carbazoles/pharmacology , DNA/drug effects , Glucosides/pharmacology , Indoles , Algorithms , Anti-Bacterial Agents/pharmacology , Calorimetry , Enzyme Inhibitors/pharmacology , Isomerism , Models, Molecular , Molecular Conformation , Oligonucleotides/chemical synthesis , Oligonucleotides/pharmacology , Surface Plasmon Resonance , Thermodynamics , Topoisomerase I Inhibitors
14.
J Biomol Struct Dyn ; 19(3): 505-13, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11790148

ABSTRACT

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.


Subject(s)
Acridines/metabolism , Bridged-Ring Compounds/metabolism , DNA/metabolism , Enzyme Inhibitors/metabolism , Telomerase/metabolism , Acridines/chemistry , Binding Sites , Bridged-Ring Compounds/chemistry , Cytosine/chemistry , DNA/chemistry , DNA, Single-Stranded/chemistry , Dimerization , Enzyme Inhibitors/chemistry , Fluorescence , Fluorescent Dyes/metabolism , G-Quadruplexes , Guanine/chemistry , Humans , Kinetics , Ligands , Nucleic Acid Conformation , Oligonucleotides/chemistry , Rhodamines/metabolism , Spectrometry, Fluorescence/methods , Telomere/chemistry , Temperature
15.
J Med Chem ; 43(24): 4711-20, 2000 Nov 30.
Article in English | MEDLINE | ID: mdl-11101362

ABSTRACT

Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials. Their anticancer activities are associated with their capacities to interact with DNA and to inhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazole chromophore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar residue contributes significantly to the DNA binding free energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycin derivatives in the drug-DNA complex remains poorly understood. To better understand how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on the sugar residue. We show that the presence of the 2'-amino function permits the formation of covalent drug-DNA complexes in the presence of formaldehyde. Complementary biochemical and spectroscopic measurements attest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing only A.T base pairs. The covalent binding to A.T base pairs was detected using a gel mobility shift assay and was independently confirmed by thermal denaturation studies and by fluorescence measurements using a series of synthetic polynucleotides. The HCHO-mediated alkylation reaction of the drug with A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs implicates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch grooves in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approach for probing minor versus major groove interactions between small molecules and DNA.


Subject(s)
Aminoglycosides , Anti-Bacterial Agents/chemical synthesis , Antibiotics, Antineoplastic/chemical synthesis , Antineoplastic Agents, Alkylating/chemical synthesis , Carbazoles/chemical synthesis , Cross-Linking Reagents/chemistry , DNA/chemistry , Formaldehyde/chemistry , Indoles , Alkylation , Anti-Bacterial Agents/chemistry , Antibiotics, Antineoplastic/chemistry , Antineoplastic Agents, Alkylating/chemistry , Carbazoles/chemistry , Daunorubicin/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorescence , Nucleic Acid Denaturation
16.
Proc Natl Acad Sci U S A ; 97(22): 12032-7, 2000 Oct 24.
Article in English | MEDLINE | ID: mdl-11027298

ABSTRACT

The binding interactions of (-)-daunorubicin (WP900), a newly synthesized enantiomer of the anticancer drug (+)-daunorubicin, with right- and left-handed DNA, have been studied quantitatively by equilibrium dialysis, fluorescence spectroscopy, and circular dichroism. (+)-Daunorubicin binds selectively to right-handed DNA, whereas the enantiomeric WP900 ligand binds selectively to left-handed DNA. Further, binding of the enantiomeric pair to DNA is clearly chirally selective, and each of the enantiomers was found to act as an allosteric effector of DNA conformation. Under solution conditions that initially favored the left-handed conformation of [poly(dGdC)](2), (+)-daunorubicin allosterically converted the polynucleotide to a right-handed intercalated form. In contrast, under solution conditions that initially favored the right-handed conformation of [poly(dGdC)](2), WP900 converted the polynucleotide to a left-handed form. Molecular dynamics studies by using the amber force field resulted in a stereochemically feasible model for the intercalation of WP900 into left-handed DNA. The chiral selectivity observed for the DNA binding of the daunorubicin/WP900 enantiomeric pair is far greater than the selectivity previously reported for a variety of chiral metal complexes. These results open a new avenue for the rational design of potential anticancer agents that target left-handed DNA.


Subject(s)
DNA/metabolism , Daunorubicin/metabolism , Allosteric Regulation , Base Sequence , Daunorubicin/chemistry , Models, Molecular , Stereoisomerism
19.
Biochemistry ; 39(29): 8439-47, 2000 Jul 25.
Article in English | MEDLINE | ID: mdl-10913249

ABSTRACT

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.


Subject(s)
DNA/chemistry , DNA/drug effects , Intercalating Agents/pharmacology , Animals , Base Sequence , Calorimetry , Cattle , DNA/metabolism , In Vitro Techniques , Models, Molecular , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Thermodynamics
20.
FEBS Lett ; 470(3): 355-9, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10745096

ABSTRACT

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.


Subject(s)
Antineoplastic Agents/metabolism , Carbazoles/metabolism , DNA/chemistry , DNA/metabolism , Glucosides/metabolism , Topoisomerase I Inhibitors , Animals , Antineoplastic Agents/chemistry , Binding, Competitive , Carbazoles/chemistry , Cattle , DNA/genetics , DNA Topoisomerases, Type I/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Dialysis , G-Quadruplexes , Glucosides/chemistry , Hot Temperature , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Nucleic Acid Conformation , Nucleic Acid Denaturation , Substrate Specificity , Thermodynamics , Ultraviolet Rays
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