PubMed Health⌕ Search

Biomedical subjects

T R Krugh

Publications and source records attributed to T R Krugh.

At least 19 recordsLinked to original sources
PubMed2001

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU).

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.

Base Sequence↗
PubMed2000

Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2).

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.

Adenosine↗
PubMed1998

Solution structure of a DNA duplex containing a replicable difluorotoluene-adenine pair.

A nonpolar aromatic nucleoside derivative based on 2,4-difluorotoluene (F), a non-hydrogen bonding shape analog of thymidine, was recently shown to be replicated against adenine with high efficiency and fidelity. This led to the suggestion that geometric matching, potentially even in the absence of hydrogen bonding between bases in a pair, may be sufficient to direct nucleotide selection during replication. We have examined the solution structure of the F-A pair in the context of a 12 base pair DNA duplex. We find that, despite the destabilization caused by this analog, the F-A pair very closely resembles that of a T x A pair in the same context. This lends support to the importance of shape matching in replication.

Adenine↗
PubMed1996

Structural features of a six-nucleotide RNA hairpin loop found in ribosomal RNA.

The hairpin loop GUAAUA occurs frequently in ribosomal RNA. Optical melting studies show that r(GGCGUAAUAGCC) folds into a hairpin containing this loop. The structural features of the r(GGCGUAAUAGCC) hairpin have been determined by NMR and molecular modeling. NOEs from G4-H1' to A9-H2 and from A9-H2 to G10-H1' show that G4 and A9 form a sheared base pair with two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3. One-dimensional NOE data show no NOEs between the imino protons of U5 and U8, but NOEs are observed between the U5-H1' and the U8-H6 and U8-H5, thus orienting the U8 imino proton away from U5. Thus U5 and U8 do not form an imino hydrogen-bonded U.U pair. The U5-H2' exhibits NOEs to both the A6-H8 and A7-H8, and the 3' phosphorus resonances of U5 and A6 are shifted downfield. This suggests that the helix turn is between the U5 and A6 nucleotides. The JH1'-H2 and JH3'-H4' coupling constants indicate that the loop is dynamic, particularly at 35 degrees C, well below the melting temperature of 63 degrees C. Structures were generated using 75 distance and 46 dihedral angle restraints. In these structures, the U5 base is stacked on the sheared base pair formed by G4 and A9 and can initiate a uridine turn similar to that observed in the anticodon loop of tRNA. The A6, A7, and U8 bases can stack on one another with their hydrogen-bonding surfaces exposed to the solvent, suggesting that they are available for tertiary interactions or protein recognition in rRNA. A range of loop structures are consistent with the data, however. The lack of formation of a U.U mismatch is consistent with a recent model that predicts the stability of hairpin loops of six nucleotides on the basis of the closing base pair and first mismatch in the loop [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289-14296].

Base Composition↗
PubMed1995

Structural characterization of a (+)-trans-anti-benzo[a]pyrene-DNA adduct using NMR, restrained energy minimization, and molecular dynamics.

The (+)-trans-anti-benzo[a]pyrene adduct formed at the N2 amino group of guanine is the major adduct found after metabolic activation of the ubiquitous carcinogen benzo[a]pyrene. The carcinogenic and mutagenic properties of the (+)-trans-anti-BP adduct, as well as related adducts, have been extensively studied. A DNA duplex containing a (+)-trans-anti-benzo[a]pyrene adduct covalently attached to the G8 nucleotide in the sequence d(CCTATGT[BP-G]CAC).d(GTGCACATAGG) was synthesized and the structure characterized by one- and two-dimensional NMR spectroscopy, in conjunction with energy minimization and molecular dynamics. This BP-11-mer duplex exhibits NOESY cross-peaks between benzo[a]pyrene protons and BP-G8, C9, A16, and C17 nucleotide protons that clearly delineate the location of the BP moiety in the minor groove of a B-type duplex with the pyrene ring oriented toward the 5' end of the modified strand. Large upfield shifts of A16 and C17 sugar resonances in the partner strand show that the pyrene moiety is situated near these sugars. Analysis of the spectra was complicated by the presence of chemical exchange line broadening of protons located near the (...T[BP-G]C...).(...GCA...) adduct site which shows the presence of a minor conformation for this BP-modified duplex in which TA is the 5' neighboring base pair. Distance restraints determined from NOESY spectra recorded at 20 degrees C were used in restrained and unrestrained energy minimization and molecular dynamics simulations to obtain a structure characteristic of the predominant conformation of the BP-11-mer duplex. The important structural features of the BP-11-mer are similar to those reported by Cosman et al. [(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918] for a (+)-trans-anti-BP adduct at a (...C[BP-G]C...).(...GCG...) sequence in which CG is the 5' neighboring base pair. No evidence of a conformational equilibrium was reported in this duplex, from which we conclude that the presence of a 5' TA base pair plays a role in the conformational equilibrium. Watson-Crick base pairing is retained in the predominant conformer of the (+)-trans-anti-BP modified duplex, which provides a visualization of a structure that could allow faithful replication. The exchange rate could not be slowed sufficiently to allow individual distance parameters to be obtained for the minor conformer.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1994

Structural characterization of two interchangeable conformations of a 2-aminofluorene-modified DNA oligomer by NMR and energy minimization.

One- and two-dimensional NMR spectroscopy and energy minimization calculations were used to investigate the conformation of a 2-aminofluorene- (AF-) modified model human c-H-ras1 protooncogene codon 61 deoxyoligonucleotide duplex, d(C1-A2-C3-C4-A5- [AF-G6]-G7-A8-A9-C10).-d(G11-T12-T13-C14-C15-T16 -G17-G18-T19-G20), in which the AF adduct is located at the third base of codon 61 with cytosine as the complementary nucleotide. Two interchangeable conformations of the AF-modified duplex, referred to as the external-AF conformation and the inserted-AF conformation, were determined from the NMR data. An analysis of the coalescence of resonances led to the estimation that the chemical exchange lifetime is greater than 3 ms but less than 20 ms at 30 degrees C, pH 7. In the external-AF conformation, Watson-Crick base-pair formation is observed for all 10 complementary nucleotides, including the AF-G6.C15 base pair. In the inserted-AF conformation, 9 of the 10 complementary bases form Watson-Crick base pairs; the AF-G6 imino proton exhibits no evidence of hydrogen bond formation with its complementary cytosine. Several NOEs between aminofluorene protons and DNA protons show that the AF moiety in the inserted-AF conformation stacks between the adjacent A5.T16 and G7.C14 base pairs. Solvated energy minimization calculations using distance restraints obtained from NOESY data at 2 degrees C with a 100-ms mixing time were performed to obtain representative structures of the external-AF and inserted-AF conformations. The external-AF conformer has the AF moiety protruding out of the major groove of a relatively unperturbed DNA duplex, leaving intact Watson-Crick base pairing for the AF-G6.C15 bases. Thus, the external-AF conformer may represent a visualization of a conformation that allows faithful replication. The inserted-AF conformer has the AF moiety stacked within the DNA helix, breaking the Watson-Crick base pairing of the modified guanine and its complementary cytosine and displacing the guanine and cytosine into the grooves. We label the inserted-AF conformer as a premutagenic conformation to reflect the displacement of the modified guanine. Interconversion between the structurally distinct external-AF and inserted-AF conformers takes place on a time scale of the same order as DNA replication. We have labeled this interconversion as a mutagenic switch to highlight a possible conformational equilibrium that may be important in replication.

Base Sequence↗
PubMed1994

2-Aminofluorene modified DNA duplex exists in two interchangeable conformations.

One- and two-dimensional NMR shows that the carcinogen 2-aminofluorene exists in two unique, interchangeable conformations when covalently bound to a model human c-H-ras1 proto-oncogene codon 61 oligomer duplex. In one conformation the 2-aminofluorene moiety protrudes out of the major groove leaving the Watson-Crick base pairing of the cytosine and 2-aminofluorene-guanine bases intact, consistent with the ability of replicating enzymes to bypass the lesion and correctly incorporate cytosine. The second form of the modified oligomer duplex may be representative of a pre-mutagenic conformation in that the 2-aminofluorene moiety is stacked within the DNA helix, disrupting base pairing between the 2-aminofluorene-modified guanine and its complementary cytosine.

Base Sequence↗
PubMed1993

Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics.

An N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex, d(C1-C2-A3-C4-[AAF-G5]-C6-A7-C8-C9).d(G10-G11-T12-G13-C14-++ +G15-T16-G17-G18), was studied by one- and two-dimensional NMR spectroscopy. Eight of the nine complementary nucleotides form Watson-Crick base pairs, as shown by NOEs between the guanine imino proton and cytosine amino protons for G.C base pairs or by an NOE between the thymine imino proton and adenine H2 proton for A.T base pairs. The AAF-G5 and C14 bases show no evidence of complementary hydrogen bond formation to each other. The AAF-G5 base adopts a syn conformation, as indicated by NOEs between the G5 imino proton and the A3-H3' and A3-H2'/H2" protons and by NOEs between the fluorene-H1 proton of AAF and the G5-H1' or C6-H1' proton. The NOEs from the C4-H6 proton to C4 sugar protons are weak, and thus the glycosidic torsion angle in this nucleotide is not well defined by these NMR data. The remaining bases are in the anti conformation, as depicted by the relative magnitude of the H8/H6 to H2' NOEs when compared to the H8/H6 to H1' NOEs. The three base pairs on each end of the duplex exhibit NOEs characteristic of right-handed B-form DNA. Distance restraints obtained from NOESY data recorded at 32 degrees C using a 100-ms mixing time were used in conformational searches by molecular mechanics energy minimization studies. The final, unrestrained, minimum-energy conformation was then used as input for an unrestrained molecular dynamics simulation. Chemical exchange cross peaks are observed, and thus the AAF-9-mer exists in more than a single conformation on the NMR time scale. The NMR data, however, indicate the presence of a predominant conformation (> or = 70%). The structure of the predominant conformation of the AAF-9-mer shows stacking of the fluorene moiety on an adjacent base pair, exhibiting features of the base-displacement [Grunberger, D., Nelson, J. H., et al. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 488-494] and insertion-denaturation models [Fuchs, R.P.P., & Daune, M. (1971) FEBS Lett. 14, 206-208], while the distal ring of the fluorene moiety protrudes into the minor groove.

2-Acetylaminofluorene↗
PubMed1990

Large-scale purification of synthetic oligonucleotides and carcinogen-modified oligodeoxynucleotides on a reverse-phase polystyrene (PRP-1) column.

A procedure is described for the large-scale purification of synthetic oligonucleotides using a polystyrene (PRP-1, Hamilton Co.) high-performance liquid chromatography (HPLC) column with a phosphate/methanol/acetonitrile solvent system. Pure oligonucleotides are obtained with a three-step procedure that involves only one column purification step. The dimethoxytrityl group is left on the oligomer for the HPLC purification. The use of the PRP-1 polystyrene column with a phosphate/methanol/acetonitrile solvent system provides excellent separation of the desired dimethoxytrityl-bearing oligonucleotide from failure sequences. The dimethoxytrityl group is removed by treatment with acetic acid and the oligonucleotide is desalted on a C-18 Sep-Pak cartridge. The oligodeoxynucleotides obtained are shown to be essentially pure by HPLC, polyacrylamide gel electrophoresis, and 500-MHzNMR spectroscopy. This procedure is especially useful for the large-scale purification of oligonucleotides required for NMR studies. The PRP-1 column and the phosphate/methanol/acetonitrile solvent system is useful for purifying modified oligonucleotides containing lipophilic groups such as the carcinogen 2-(acetylamino)fluorene.

Base Sequence↗
PubMed1989

Two-dimensional NMR studies on the anthramycin-d(ATGCAT)2 adduct.

Two-dimensional NMR experiments were performed on the adduct of anthramycin with d(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATamGCAT).d(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOEs to several nucleotide protons. The network of anthramycin-nucleotide NOEs and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H11a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The NOE data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the J1'-2' coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred.

Anthramycin↗
PubMed1986

The antitumor agent mitoxantrone binds cooperatively to DNA: evidence for heterogeneity in DNA conformation.

The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione], to various DNAs has been examined by optical titration and equilibrium dialysis methods. At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs. The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding. In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values. At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process. A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms. The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process. This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA. It is suggested that clustering may play a role in the physiological activity of drugs.

Animals↗
PubMed1986

Fluorescence-detected circular dichroism of ethidium bound to poly(dG-dC) and poly(dG-m5dC) under B- and Z-form conditions.

The equilibrium binding of ethidium to poly(dG-dC) and poly(dG-m5dC) under conditions favoring B and Z forms was investigated with fluorescence-detected circular dichroism (FDCD) and optical titration methods. FDCD spectra indicate a similar geometry for the intercalated ethidium under both B- and Z-form conditions, even at low levels of bound ethidium. The magnitude of the 310-330-nm FDCD band as a function of the bound drug to base pair ratio (r) indicates ethidium binds to poly(dG-dC) in 4.4 M NaCl and to poly(dG-m5dC) in 25 mM MgCl2 by clustering. Under these conditions, circular dichroism spectra indicate the polymer is largely Z form. Thus, it appears ethidium clusters into regions it has induced into a right-handed form. For all conditions studied, the FDCD spectra provided no evidence for a left-handed binding site. Under B-form conditions, binding is random.

Chemical Phenomena↗
PubMed1985

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution.

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthramycin↗
PubMed1985

Ethidium binding to left-handed (Z) DNAs results in regions of right-handed DNA at the intercalation site.

The equilibrium binding of ethidium to the right-handed (B) and left-handed (Z) forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) was investigated by optical and phase partition techniques. Ethidium binds to the polynucleotides in a noncooperative manner under B-form conditions, in sharp contrast to highly cooperative binding under Z-form conditions. Correlation of binding isotherms with circular dichroism (CD) data indicates that the cooperative binding of ethidium under Z-form conditions is associated with a sequential conversion of the polymer from a left-handed to a right-handed conformation. Determination of bound drug concentrations by various titration techniques and the measurement of circular dichroism spectra have enabled us to calculate the number of base pairs of left-handed DNA that adopt a right-handed conformation for each bound drug; 3-4 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to the right-handed form for each bound ethidium, while approximately 25 and 7 base pairs switch conformations for each bound ethidium in complexes with poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2, respectively. The induced ellipticity at 320 nm for the ethidium-poly(dG-dC).poly(dG-dC) complex in 4.4 M NaCl indicates that the right-handed regions are nearly saturated with ethidium even though the overall level of saturation is very low. The circular dichroism data indicate that ethidium intercalates to form a right-handed-bound drug region, even at low r values where the CD spectra show that the majority of the polymer is in a left-handed conformation.

Chemical Phenomena↗
PubMed1985

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix.

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1985

N-acetoxy-2-acetylaminofluorene modification of a deoxyoligonucleotide duplex.

The carcinogen N-acetoxy-2-acetylaminofluorene was reacted with d (CCACGCACC) to form a covalent adduct with attachment at the single guanine. The sample was purified, mixed 1:1 with d (GGTGCGTGG) and studied by thermal denaturation experiments. The Tm for the mixture was 35 +/- 3 degrees C, consistent with duplex formation. The method of continuous variation shows that the modified oligomer, d (CCACGAAFCACC), forms a 1:1 duplex with d (GGTGCGTGG). Circular dichroism spectra also indicate the formation of a duplex and suggest that the modified duplex has a left-handed conformation. Addition of the intercalating drug ethidium alters the CD spectrum of the modified duplex, resulting in a CD spectrum similar to that of ethidium bound to right-handed DNA.

2-Acetylaminofluorene↗
PubMed1984

The reaction of anthramycin with DNA. Proton and carbon nuclear magnetic resonance studies on the structure of the anthramycin-DNA adduct.

Nuclear magnetic resonance techniques are used to confirm the points of attachment of anthramycin to DNA. Using 13C NMR spectroscopy, the C-11 resonance of anthramycin is shown to undergo a 16-ppm upfield shift upon formation of a covalent bond with DNA, indicative of an aminal linkage at that position. The site of attachment on the DNA is determined using the self-complementary oligodeoxyribonucleotide d-(ApTpGpCpApT) as a DNA model. Proton NMR, both in H2O and D2O solutions, provides a direct characterization of the anthramycin-oligonucleotide adduct. Upon covalent attachment to the duplex, a loss in the helical symmetry is observed, resulting in a doubling of several of the oligonucleotide resonances. Examination of the data confirms that the point of attachment of the anthramycin to the d-(ApTpGpCpApT) is at the guanine-NH2-position, consistent with the model proposed by Hurley and Petrusek (Hurley, L. H., and Petrusek, R. L. (1979) Nature (Lond.) 282, 529-531) and Petrusek et al. (Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., and Hurley, L. H. (1981) Biochemistry 20, 1111-1119).

Animals↗