Drug‐DNA binding specificity: Binding of netropsin and distamycin to poly(d2NH 2 A‐dT)

The interaction of the minor groove binding drug distamycin with the T-A-T triple helix and the A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3'-phosphate of one strand and the 5'-phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide to be in a duplex form with a dangling arm. Circular dichroism spectroscopy demonstrates that the distamycin A molecule can bind to the triple-stranded form of this oligonucleotide. Spectral analysis shows that the bound distamycin exhibits a conformation and an environment slightly different from those which are observed when the drug is bound to the corresponding double-stranded structure. Furthermore, a second type of complex which is observed in the double-strand binding (two stacked distamycins in the minor groove) is not observed with the triple-stranded host. When distamycin is added to the triplex made of unbridged chains (dA)12 + 2(dT)12, the triplex dissociates to give a double-stranded structure. Thermal denaturation experiments demonstrate that distamycin binding destabilizes the triplex whereas it stabilizes the duplex. These results are compared with those obtained by the same experimental approaches on other minor groove binding drugs.

Section: Resultsmentioning

confidence: 99%

Distamycin A Complexation with a Nucleic Acid Triple Helix

Durand

Maurizot

1996

“…Crystallographic studies also show that distamycin A preferentially binds to double-helical DNA in the B-form with C2'-endo sugar pucker and not in the A-form with C3'-endo sugar pucker (Kopka et al, 1985;Coll et al, 1989). This specificity is attributed to maximum contacts between the sugar phosphate backbone of the duplex DNA of B-form geometry and distamycin A (Coll et al, 1989;Dasgupta et al, 1990). In exploring the interaction of distamycin (Dst) with doublehelical (Dasgupta et al, 1990) and triple-helical polynucleotides, we observed that distamycin binds to the triple helix d(T)"'d(A)"'d(T)" (see below).…”

mentioning

confidence: 94%

“…This specificity is attributed to maximum contacts between the sugar phosphate backbone of the duplex DNA of B-form geometry and distamycin A (Coll et al, 1989;Dasgupta et al, 1990). In exploring the interaction of distamycin (Dst) with doublehelical (Dasgupta et al, 1990) and triple-helical polynucleotides, we observed that distamycin binds to the triple helix d(T)"'d(A)"'d(T)" (see below). Since this triple helix has been characterized as A-form DNA, the unexpected binding result caused us to examine further the conformation of this helix.…”

mentioning

confidence: 99%

Structure of d(T)n.cntdot.d(A)n.cntdot.d(T)n: the DNA triple helix has B-form geometry with C2'-endo sugar pucker

Howard¹,

Miles²,

Liu³

et al. 1992

Self Cite

The polynucleotide helix d(T)n.d(A)n.d(T)n is the only deoxypolynucleotide triple helix for which a structure has been published, and it is generally assumed as the structural basis for studies of DNA triplexes. The helix has been assigned to an A-form conformation with C3'-endo sugar pucker by Arnott and Selsing [1974; cf. Arnott et al. (1976)]. We show here by infrared spectroscopy in D2O solution that the helix is instead B-form and that the sugar pucker is in the C2'-endo region. Distamycin A, which binds only to B-form and not to A-form helices, binds to the triple helix without displacement of the third strand, as demonstrated by CD spectroscopy and gel electrophoresis. Molecular modeling shows that a stereochemically satisfactory structure can be build using C2'-endo sugars and a displacement of the Watson-Crick base-pair center from the helix axis of 2.5 A. Helical constraints of rise per residue (h = 3.26 A) and residues per turn (n = 12) were taken from fiber diffraction experiments of Arnott and Selsing (1974). The conformational torsion angles are in the standard B-form range, and there are no short contacts. In contrast, we were unable to construct a stereochemically allowed model with A-form geometry and C3'-endo sugars. Arnott et al. (1976) observed that their model had short contacts (e.g., 2.3 A between the phosphate-dependent oxygen on the A strand and O2 in the Hoogsteen-paired thymine strand) which are generally known to be outside the allowed range.(ABSTRACT TRUNCATED AT 250 WORDS)

“…The preference of NET and DST binding to A‚T sequences was further rationalized in terms of the protrusion of the amino group of guanosine into the minor groove which interferes sterically with binding at sites containing G‚C pairs (4). This assertion, however, was being challenged by a study with poly(d2NH 2 A-dT) indicating that despite the presence of the 2-amino group this polynucleotide exhibits significant binding to NET and DST (5).…”

mentioning

confidence: 99%

Circular Dichroic and Kinetic Differentiation of DNA Binding Modes of Distamycin

Chen

Sha

1998

DNA binding modes of distamycin (DST) were investigated via comparative binding studies with oligomeric duplexes of the form d(GCG-X-GCG).d(CGC-Y-CGC), where Y is complementary to X and X = 4- or 5-base binding site. It was found that 1:1 and 2:1 drug-duplex complexes exhibit distinctly different circular dichroic (CD) spectral characteristics and can, thus, serve as diagnostic tools for binding mode differentiation. CD intensity profiles at 265 or 275 nm as a function of drug to DNA ratios can reveal the extent of binding cooperativity for 2:1 complex formation (i.e., the relative binding affinities of 2:1 vs 1:1) at a 5-base-paired binding site. Comparison of these profiles leads to the following qualitative ranking for the binding cooperativity for the studied sites: AAGTT, ATATA >/= AAACT > AATAA, AAATA, AAAGT > AATAT > TAAAA >/= AAATT >/= AAAAA >/= ATAAA, AAAAT. The plausibility of this ordering is strengthened by its agreement with the ranking established by earlier NMR studies on some of the sequences. The significantly slower DST dissociation kinetics of the 2:1 complexes as compared to those of 1:1 made the kinetic measurements of SDS-induced dissociation by the stopped-flow technique possible. The results indicate that the AAGTT site exhibits the slowest DST dissociation rate, with a characteristic time of 35 s. The rates of dissociation in general correlate reasonably well with the cooperativity order found via equilibrium CD measurements (the higher the binding cooperativity, the slower the rate of dissociation). Base sequence specific binding of DST was also found for the 1:1 complex formation at the 4-base-paired sites, with AAAA, TTTT, ATTT, and AAAT sequences exhibiting the highest binding affinities.