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PubMedDate not supplied

[Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I].

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.

Base Sequence↗

PubMed1985

Netropsin specifically recognizes one of the two conformationally equivalent strands of poly(dA).poly(dT). One dimensional NMR study at 500 MHz involving NOE transfer between netropsin and DNA protons.

Recent observations that the heteronomous structural model for poly(dA).poly(dT) is not found in solution and that in this DNA, the two strands are conformationally equivalent (J. Biomole. Str. Dyns. 2, 1057 (1985], has added a new dimension to the structural dynamics of DNA-netropsin complex. Does the antibiotic somehow distinguish between the two strands and specifically interact with only one of the conformationally equivalent strands? Model-building studies suggest that netropsin can either bind to the dA-strand in the minor groove such that H-bonds are formed between the imino protons N4-H, N6-H, N8-H of netropsin and N3 atoms of A or can bind to the dT-strand in the minor groove and form H-bonds between the imino-protons N4-H, N6-H, N8-H of netropsin and O2 atoms of T. If netropsin binds to the dA-strand, AH2 atoms of poly(dA).poly(dT) would be in closer proximity to the imino protons N4-H, N6-H, N8-H and pyrrole ring protons C5-H, C11-H of netropsin than they would be, if netropsin binds to the dT-strand. In order to distinguish these possibilities experiments were conducted which involved NOE energy transfer between netropsin and DNA protons in the drug-DNA complex. Difference NOE spectra of netropsin-poly(dA).poly(dT) complex in which AH2 was irradiated indicate that dominant NOEs were observed at the imino and pyrrole ring protons of netropsin. When the netropsin pyrrole ring protons were irradiated, the magnetization transfer was at AH2 of DNA. These observations suggest that netropsin binds to the dA-strand of poly(dA).poly(dT) even though dA/dT strands are conformationally equivalent.

Guanidines↗

PubMedDate not supplied

[Ligands with specific affinity to a specific sequence of DNA base pairs. XI. The synthesis and binding to DNA of bis-netropsins with the C-ends of their netropsin fragments tethered by tetra- or pentamethylene linkers].

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A.T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5'-A4T(4)-3' sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5'-T4A(4)-3' sequences. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.

Base Pairing↗

PubMed1991

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.

Animals↗

PubMedDate not supplied

[Netropsin, distamycin A, bis-netropsins as selective inhibitors of restrictases and DNAse I].

The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netropsin and bis-netropsins show different nucleotide sequence specificity. This fact can be used for selective inhibition of restriction endonucleases.

DNA↗

PubMed2002

Structure of the 1:1 netropsin-decamer d(CCIICICCII)2 complex with a single bound netropsin.

The crystal structure of the 1:1 complex of netropsin and the B-DNA decamer d(CCIICICCII)(2) has been elucidated and refined to an R factor of 19.6% and an R(free) of 24.7% using 1790 reflections in the resolution range 8-2.4 A. The complex crystallizes in space group C2, with unit-cell parameters a = 62.40, b = 24.47, c = 36.31 A, beta = 110.09 degrees and one molecule of netropsin in the asymmetric unit; the rest of the minor groove is filled with six water molecules. The structure was solved by the molecular-replacement method using the DNA model d(CCCCCIIIII)(2) from the 2:1 netropsin complex by removing both bound netropsins (Chen et al., 1998). Surprisingly, only one netropsin molecule is found to bind to the present decamer, covering residues 2-6 at the upper stream of the duplex. The positively charged guanidinium head is hydrogen bonded through N1H(2) to the O(2) of cytosine 2 and through N10H(2) to N(3) of inosine 6. The three amide N-H groups of the peptides face the minor groove and form three sets of bifurcated hydrogen bonds with the base atoms. The central part of the drug (C3-N8) is nearly conjugated. The preference of the cytosine carbonyl O2 atoms over the inosine N3 atoms in hydrogen bonding is seen. The drug-bound region has more uniform twists, roll angles, propeller twists and minor-groove widths compared with the water-bound region.

Anti-Bacterial Agents↗

PubMed1977

Netropsin-poly(dA-dT) complex in solution: structure and dynamics of antibiotic-free base pair regions and those centered on bound netropsin.

The biphasic duplex-to-strand transition for the netropsin.poly(dA-dT) complex, phosphate/drug mole ratio (P/D) = 50, has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectroscopy at the nonexchangeable base and sugar protons in 0.1 M cacodylate solution. The NMR spectral parameters monitor the structure and dynamics of the opening of antibiotic-free base pair regions (55 degrees-65 degrees) and the opening of base regions centered on bound netropsin (90 degrees-100 degrees). The gradual addition of netropsin to poly(dA-dT) results in structural perturbations extending into the antibiotic-free base pair regions that begin to level off above 0.02 antibiotic molecules per polynucleotide phosphate (P/D = 50). The NMR chemical shift parameters at the antibiotic-free base pair regions in the P/D = 50 complex suggest changes in the glycosidic torsion angles of the deoxyadenosine and thymidine residues and less pronounced changes in the base pair overlap geometries. The dissociation rates of the antibiotic-free base pair regions are at least an order of magnitude slower in the P/D = 50 netropsin.poly(dA-dT) complex compared to related parameters for poly(dA-dT) and the P/D = 50 ethidium bromide-poly(dA-dT) complex. There is decreased segmental mobility at the antibiotic-free strand regions in the temperature range (65 degrees-90 degrees) between the two transitions in the biphasic melting curve of the P/D = 50 netropsin-poly(dA-dT) complex. Netropsin stabilizes at least five base pairs, with their center at its binding site.

Guanidines↗

PubMed1982

Specific interaction of netropsin, distamycin-3 and analogs with LC duplexes: reversion towards the B form of the 2'-deoxy-.2'-deoxy-2'-fluoro-hybrid duplexes upon specific interaction with netropsin, distamycin-3 and analogs.

Binding of the B-form specific ligands netropsin and distamycin-3, -4 and -5 has been used to monitor the presence and/or the inducibility of a B-type structure in various poly-inosinic.poly-cytidilic double stranded polymers with deoxyribose, ribose or 2'-deoxy-2'-fluororibose as sugar on either strand. The efficiency of binding was followed by circular dichroism and further evaluated by the increase in melting temperature of the complexes. The efficient binding of netropsin and distamycins to the hybrid polymer (dIfl)n. (dC)n demonstrated that the fluorine carrying strand may undergo a A to B-type transition reflecting a change of the 2'-deoxy-2'-fluororibose from the 3'-endo to the 1'-exo or 2'-endo pucker. The less efficient binding of the same ligands to the reverse hybrid (dI)n.(dCfl)n showed that the geometry of the pyrimidine strand is the most critical for the specific interaction. Taking into account the recent findings about the regular hydration in the minor groove of the B-type dodecamer dCGCGAATTCGCG in solid-state, the different binding modes observed between the different polymers and antibiotics are explained by differences in their possibilities of hydration. Binding of netropsin to a double stranded deoxypolymer is interpreted as a local replacement of water molecules by netropsin in the minor groove hydration network which is typical of the B-form.

Chemical Phenomena↗

PubMed1993

Inhibition of the Moloney murine leukemia virus cycle at a post reverse transcriptional step by the netropsin-intercalating hybrid molecule netropsin-oxazolopyridocarbazole.

In a search for new antiretroviral agents acting at the nucleic acid level, two hybrid molecules composed of a bispyrrolecarboxamide chain related to netropsin, linked to the intercalating chromophore oxazolopyridocarbazole, were tested on the cycle of a defective Moloney murine leukemia virus (M.MuLV) derived from the SVX shuttle and expressing resistance to the G418 antibiotic. The drug netropsin-oxazolopyridocarbazole (Net-OPC), which displays a binding preference to duplex DNA containing A + T bases, inhibits the retroviral replicative cycle (IC50 = 4.8 microM). In contrast, the related molecule (bis)pyrollecarboxamide-oxazolopyridocarbazole (Bpc-OPC) devoid of sequence preference as well as the elemental components of Net-OPC, namely OPC, pentyl-OPC and netropsin, displays no significant action on the viral cycle. The estimation of cytosolic viral DNA in infected cells using quantitative polymerase chain reaction suggests that Net-OPC impairs a post retrotranscriptional step of the viral cycle.

3T3 Cells↗

PubMed1978

Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex.

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.

Animals↗

PubMed1992

Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.

Calorimetry, Differential Scanning↗

PubMed1994

Influence of drug binding on DNA hydration: acoustic and densimetric characterizations of netropsin binding to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes and the poly(dT).poly(dA).poly(dT) triplex at 25 degrees C.

We use high-precision acoustic and densimetric techniques to determine, at 25 degrees C, the changes in volume, delta V, and adiabatic compressibility, delta Ks, that accompany the binding of netropsin to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes, as well as to the poly(dT).poly(dA).poly(dT) triplex. We find that netropsin binding to the heteropolymeric poly(dAdT).poly(dAdT) duplex is accompanied by negative changes in volume, delta V, and small positive changes in compressibility, delta Ks. By contrast, netropsin binding to the homopolymeric poly(dA).poly(dT) duplex is accompanied by large positive changes in both volume, delta V, and compressibility, delta Ks. Furthermore, netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes changes in both volume and compressibility that are nearly twice as large as those observed when netropsin binds to the poly(dA).poly(dT) duplex. We interpret these macroscopic data in terms of binding-induced microscopic changes in the hydration of the DNA structures and the drug. Specifically, we find that netropsin binding induces the release of approximately 22 waters from the hydration shell of the poly(dAdT).poly(dAdT) heteropolymeric duplex, approximately 40 waters from the hydration shell of the poly(dA).poly(dT) homopolymeric duplex, and about 53 waters from the hydration shell of the poly(dA).poly(dT), induces the release of 18 more water molecules than netropsin binding to the heteropolymeric duplex, poly(dAdT).poly(dAdT). On the basis of apparent molar volume, phi V, and apparent molar adiabatic compressibility, phi Ks, values for the initial drug-free and final drug-bound states of the two all-AT duplexes, we propose that the larger dehydration of the poly(dA).poly(dT) duplex reflects, in part, the formation of a less hydrated poly(dA).poly(dT)-netropsin complex compared with the corresponding poly(dAdT).poly(dAdT)-netropsin complex. In conjunction with our previously published entropy data [Marky, L. A., & Breslauer, K. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4359-4363], we calculate that each water of hydration released to the bulk solvent by ligand binding contributes 1.6 cal K-1 mol-1 to the entropy of binding. This value corresponds to the average difference between the partial molar entropy of water in the bulk state and water in the hydration shells of the two all-AT duplexes. When netropsin binds to the poly(dT).poly(dA).poly(dT) triplex, the changes in both volume and compressibility suggest that the binding event induces more dehydration of the triplex than of the duplex state. Specifically, we calculate that netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes the release of 13 more waters than netropsin binding to the poly(dA).poly(dT) duplex.(ABSTRACT TRUNCATED AT 400 WORDS)

Biophysical Phenomena↗

PubMed1997

Effects of bifunctional netropsin-related minor groove-binding ligands on mammalian type I DNA topoisomerase.

We investigated the effects of compounds with two covalently linked netropsin moieties (bis-netropsin) on the function of mammalian type I DNA topoisomerase (topo I) in vitro. We initiated these studies because earlier studies had shown that certain bis-netropsins possess a several-fold higher antitumor and antiviral activity than netropsin. We confirmed that the parent compound netropsin, but not its bifunctional derivatives, induce supercoils in closed DNA. We determined that bis-netropsins inhibit the binding of topo I to DNA more efficiently than netropsin and that bis-netropsins but not netropsin induce specific DNA strand cleavage in the presence of topo I. We discuss a model explaining the different effects of netropsin and bis-netropsins on topo I.

Antineoplastic Agents↗

PubMed1984

Structure and dynamics of netropsin-poly(dA-dT).poly(dA-dT) complex: 500 MHz 1H NMR studies.

Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and O2 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA--but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above. Using poly(dA-dT).poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific H-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat. By monitoring the 31P signals of free poly(dA-dT).poly(dA-dT) and netropsin-poly(dA-dT).poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base-pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT).poly(dA-dT). This experiment was conducted in 90% H2O + 10% D2O using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D2O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT).poly(dA-dT)-netropsin complex. In summary, experimental data indicate that netropsin binds to poly(dA-dT).poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1983

Calorimetric and spectroscopic investigation of drug-DNA interactions. I. The binding of netropsin to poly d(AT).

We report the first calorimetric investigation of netropsin binding to poly d(AT). Temperature-dependent uv absorption, circular dichroism (CD), batch calorimetry, and differential scanning calorimetry (DSC) were used to detect, monitor, and thermodynamically characterize the binding process. The following results have been obtained: 1) Netropsin groove binding is accompanied by a large exothermic enthalpy of 9.2 kcal/mol of drug bound at 25 degrees C. This indicates that a large negative binding enthalpy may be a necessary but not a sufficient criterion for drug intercalation. We suggest that the exothermic binding might be correlated with specific H-bonding interactions. 2) From the difference in DSC transition enthalpies in the presence and absence of netropsin, we calculate a binding enthalpy of -10.7 kcal/mol of netropsin at 88 degrees C. 3) We calculate a positive delta S for netropsin binding to poly d(AT) at 25 degrees C. This positive entropy change may reflect netropsin-induced release of condensed cations and/or bound water. 4) The netropsin-saturated duplex monophasically melts 46 degrees C higher than the free duplex. The unsaturated duplex melts through two thermally-resolved transitions that correspond to netropsin-free and netropsin-bound regions. These two regions interact dynamically with no substantial influence on the thermal stabilities of the separate domains. 5) Netropsin binding decreases the cooperativity of the duplex to single strand transition.

Calorimetry↗

PubMedDate not supplied

[Specific DNA cleavage by an analog of netropsin containing a copper(II) chelating peptide Gly-Gly-His].

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.

Amino Acid Sequence↗

PubMed2005

A host-guest approach for determining drug-DNA interactions: an example using netropsin.

Netropsin is a well-characterized DNA minor groove binding compound that serves as a model for the study of drug-DNA interactions. Our laboratory has developed a novel host-guest approach to study drug-DNA interactions in which the host, the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is co-crystallized with a DNA oligonucleotide guest in the presence and absence of drug. We have co-crystallized netropsin with the RT fragment bound to the symmetric 16mer d(CTTAATTCGAATTAAG)2 and determined the structure of the complex at 1.85 A. In contrast to previously reported netropsin-DNA structures, our oligonucleotide contains two AATT sites that bind netropsin with flanking 5' and 3' sequences that are not symmetric. The asymmetric unit of the RT fragment-DNA-netropsin crystals contains one protein molecule and one-half of the 16mer with one netropsin molecule bound. The guanidinium moiety of netropsin binds in a narrow part of the minor groove, while the amidinium is bound in the widest region within the site. We compare this structure to other Class I netropsin-DNA structures and find that the asymmetry of minor groove widths in the AATT site contributes to the orientation of netropsin within the groove while hydrogen bonding patterns vary in the different structures.

AT Rich Sequence↗

PubMedDate not supplied

[Effect of DNA local conformation on the affinity and binding specificity of bis-netropsins to DNA].

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group (<--Nt-Pt(NH3)-Nt-->) or aliphatic pentamethylene chain (<--Nt-(CH2)5-Nt-->), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of <--Nt-(CH2)5-Nt--> with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for <--Nt-Pt(NH3)-Nt--> and <--Nt-(CH2)5-Nt--> binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.

Anti-Bacterial Agents↗