Netropsin DNA complexes

Polyamides 2 and 3, which preserve the Im-)S-Im unit, bound DNA sites of similar size, 10- and 11-base pairs, but remarkably different sequence compositions. In the original lexitropsin model based on 1 1 binding of polyamides to DNA, Im was proposed to bind GC/CG > AT/TA [1]. We now know this not to be the case. In a study to be published elsewhere, Im, in the sequence context of polyamide 2, binds all four base pairs (within a factor of 2) [19]. From the crystal structure of the 1 1 netropsin DNA complex, we understand the molecular mechanism by which Py specifies AT/TA > GC/CG [1]. However, this 1 1 recognition code of Py selecting AT/TA > GC/CG must now be modified to include the judicious placement of jS for AT/TA recognition to reset the curvature in the 1 1 motif [9]. This is substantiated by our observation that Im- -Im binds 5 -GAG-3 with higher affinity than Im-Py-Im (Table). 

Martin JC, Wartell RM, O Shea I (1978) Conformational features of distamycin-DNAand netropsin-DNA complexes by Raman spectroscopy. Proc Natl Acad Sci USA75(12) 5483-... 

The MD simulations were carried out under standard temperature and pressure. A 1 fs time step was used with SHAKE25 applied to bonds. A 2 fs time step with SHAKE was used in the d(IC)6 d(IC)6 —> d(GC)6 d(GC)6 calculations. The non-bonded interactions for DNA complexes were subject to 10 -12 A spherical cutoff whereas no cutoff was applied to solute-solute interactions to avoid cutoff artifacts on coulombic interactions between sodium ions with phosphates. In the case of d(IC)6 d(IC)6 —> d(GC)6 d(GC)6 calculations an 8 A spherical cutoff was applied to non-bonded interactions. A weak harmonic restraint of 5.0 kcal/mol was imposed to avoid the disruption of terminal base pairs during FEP simulations of netropsin —> 0 and 2-imidazole-distamycin —> distamycin calculations. 

Kopka et al. (186) solved the X-ray crystal structure of netropsin (1) complexed with CGCGAATTBrCGCG at 2.2 A resolution. Netropsin binds to the minor groove of double helical B-DNA and is selective for four or more A-T (adenine-thymine) base pairs a single G-C (guanosine-cytosine) pair prevents binding (see color insert, Figure 10). Netropsin s amide NH groups... 

The free energy calculations with complexes of oligomeric DNA with distamycin and netropsin presented in this chapter were carried out using the AMBER force field2 and the AMBER software.21 The associated potential energy function is ... 

Taquet A, Labarbe R, Houssier C (1998) Calorimetric investigation of ethidium and netropsin binding to chicken erythrocyte chromatin. Biochemistry 37(25) 9119—9126 Temple MD, McEadyen WD, Holmes RJ, Denny WA, Murray V (2000) Interaction of cisplatin and DNA-targeted 9-aminoacridine platinum complexes with DNA. Biochemistry 39(18) 5593-5599 Terasaki T, Iga T, Sugiyama Y, Hanano M (1984) Interaction of doxorubicin with nuclei isolated from rat liver and kidney. J Pharm Sci 73(4) 524—528... 

Repeat Question 11, but graph the data as a linear, double reciprocal plot in the spirit of the Lineweaver-Burk equation (see Chapter 4). Plot l/ATm vs. 1 /(N/nt) and perform a linear regression to determine the best-fit line (Equation 4.a). The x-intercept corresponds to the KD of the DNA-netropsin complex. The KD value from this method should be more accurate than the estimation in Question 11. 

In the thermodynamic study of duplex formation, a variety of complementary pairs of relatively simple, well-defined oligonucleotides are employed,73-77 while the intercalation thermodynamics was examined with more complex or natural DNA duplexes.57,59 69,78-89 Typical intercalating agents examined are acridine orange,78 acriflabine,78 actinomycin,79 daunomycin,57,59,79-81 ethidium bro-mide,59,69,78,82,83 and netropsin.59,74,79,84,85... 

We first describe the NMR parameters for the duplex to strand transition of the synthetic DNA poly(dA-dT) (18) with occasional reference to poly(dA-dU) (24) and poly(dA- brdU) and the corresponding synthetic RNA poly(A-U) (24). This is followed by a comparison of the NMR parameters of the synthetic DNA in the presence of 1 M Na ion and 1 M tetramethylammonium ion in an attempt to investigate the effect of counterion on the conformation and stability of DNA. We next outline structural and dynamical aspects of the complexes of poly(dA-dT) with the mutagen proflavine (25) and the anti-tumor agent daunomycin (26) which intercalate between base pairs and the peptide antibiotic netropsin (27) which binds in the groove of DNA. 

Netropsin complexes with nucleic acids have been monitored by spectroscopic techniques at the oligomer duplex and DNA level (89-96). Our research focussed on the application of high resolution NMR spectroscopy to elucidate structural and dynamic aspects of netropsin complexes with the self-complementary octanucleotide dG-dG-dA-dA-dT-dT-dC-dC duplex (9 7) and the synthetic DNA poly(dA-dT) (27) in aqueous solution. 

Netropsin Migration Along Partially Opened DNA The plots of the sugar H-l chemical shifts in the Nuc/D = 50 netropsin poly(dA-dT) complex in 0.1 M buffer demonstrate that the lower temperature and higher temperature cooperative transitions exhibit midpoints of 61°C and 95°C, respectively (Figure 36). This section covers the NMR spectral parameters for the complex between these temperature values (65 and 90°C) with typical spectra in the 5 to 9 ppm presented in Figure 38. 

The observation of selective complexation shifts in the nucleic acid resonances of the synthetic DNA demonstrate a change in the glycosidic torsion angles of the adenosine and thymidine residues and a minimal perturbation in the base pair overlaps on addition of netropsin. These structural perturbations at the antibiotic binding site are propagated to adjacent antibiotic-free base pair regions at low netropsin concentrations. 

We observe that netropsin binds tightly to DNA and stabilizes 5 base pairs centered about its binding site. The opening rates of the intervening base pair stretches during the dissociation of the antibiotic-free base pair regions in the Nuc/D = 50 complex are slower by an order of magnitude compared to the dissociation rates for the duplex to strand transition of poly(dA-dT) alone in 0.1 M buffer solution. 

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