Biomedical subjects

D J Patel

Publications and source records attributed to D J Patel.

At least 91 records · Page 5Linked to original sources

PubMed1996

Solution conformation of the (-)-cis-anti-benzo[a]pyrenyl-dG adduct opposite dC in a DNA duplex: intercalation of the covalently attached BP ring into the helix with base displacement of the modified deoxyguanosine into the major groove.

This paper reports on the combined NMR-molecular mechanics computational studies of the solution structure of the (-)-cis-anti-[BP]dG adduct positioned opposite dC in the sequence context d(C1- C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14- A15-G16-C17-G18-A19-T20- G21-G22) duplex [designated (-)-cis-anti-[BP]dG.dC 11-mer duplex]. This adduct is derived from cis addition at C10 of (-)-anti-7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] to the N2 position of dG6 in this duplex sequence. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and nucleic acid of the major conformation were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of proton resonances for a three-nucleotide segment centered about the lesion site which resulted in a tentative assignment for the sugar protons of the C7 residue in the spectrum of the adduct duplex. The solution conformation of the major conformation of the (-)-cis-anti-[BP]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and intermolecular BP-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the covalently attached benzo[a]pyrenyl ring intercalates between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine [BP]-dG6 and its partner cytosine dC17 are looped out of the helix into the major groove. The purine ring of the [BP]dG6 residue is directed toward the 5'-end of the modified strand and stacks over the major groove edge of its 5'-side neighbor dC5 residue. The solution structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex is compared with those of the stereoisomeric (+)-trans-anti-[BP]dG [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918], (-)-trans-anti-[BP]dG [de los Santos, C., et al. (1992) Biochemistry 31, 5245-5252], and (+)-cis-anti-[BP]dG [Cosman, M., et al. (1993a) Biochemistry 32, 4146-4155] adducts positioned opposite dC in the same duplex sequence context. A key finding is that the long axes of the intercalated benzo[a]pyrenyl rings in the solution structures of the (+)- and (-)- cis-anti-[BP]dG.dC 11-mer duplexes are oriented in opposite directions with the benzylic ring directed toward the minor groove in the (+)-cis isomer and toward the major groove in the (-)-cis isomer. In addition, a comparison is also made with the solution structure of the (+)-trans-anti-[BP]dG adduct opposite a deletion site [Cosman, M., et al. (1994a) Biochemistry 33, 11507-11517] since this adduct duplex displays several conformational features in common with the structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex. The structures of both duplex adducts exhibit intercalation of the covalently attached ligand into the helix and displacement of the modified deoxyguanosine into the major groove. Studies of the biological activities of stereochemically defined BP-DNA adducts and the comparison of the solution structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex with its stereoisomeric counterparts should lead to new insights into the relationships between defined helical distortions and mutagenic specificity and activity.

Base Composition↗

PubMed1996

Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.

The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.

Adenosine Monophosphate↗

PubMed1996

Tenuous link between ischaemic threshold and both ambulatory ischaemia and symptom status over time in stable angina: a 5-year follow-up study.

This study evaluates changes in ischaemic threshold over a 5-year period in patients with stable angina pectoris, who did not suffer any intervening cardiac event. Changes in ischaemic threshold are related to alterations in symptomatic status and ambulatory ischaemia. Over long-term follow-up, there is a significant fall in ischaemic threshold in such patients (mean heart rate at onset of ischaemia fell from 104 +/- 17.8 to 97 +/- 17.4 bpm: P < 0.001), but this is not matched by a worsening of either symptoms or ischaemia during daily life. In the 68% of patients that had a reduction in ischaemic threshold of > or = 5 bpm, 68% had either definite reduction or no change in symptoms and 84% had either reduction, abolition or no change in transient ischaemic activity. The dissociation between ischaemic threshold, ambulatory ischaemia and symptoms has implications for long-term monitoring and management of the patient with stable angina.

Adult↗

PubMed1996

Solution structure of the donor site of a trans-splicing RNA.

BACKGROUND: RNA splicing is both ubiquitous and essential for the maturation of precursor mRNA molecules in eukaryotes. The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the 22-nucleotide SL from the SL1 RNA. Very little is known about the various roles that RNA structures play in the complex conformational rearrangements and reactions involved in premRNA splicing. RESULTS: We have determined the solution structure of a domain of the first stem loop of the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMR techniques; this domain contains the splice-donor site and a nine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts a stem-loop structure: nucleotides in the stem region form a classical A-type helix while nucleotides in the hairpin loop specify a novel conformation that includes a helix, that extends for the first three residues; a syn guanosine nucleotide at the turn region; and an extrahelical adenine that defines a pocket with nucleotides at the base of the loop. CONCLUSION: The proximity of this pocket to the splice donor site, combined with the observation that the nucleotides in this motif are conserved among all nematode SL RNAs, suggests that this pocket may provide a recognition site for a protein or RNA molecule in the trans-splicing process.

Animals↗

PubMed1996

DNA bending and unwinding associated with actinomycin D antibiotics bound to partially overlapping sites on DNA.

Actinomycin D (ActD) is a potent anti-tumor antibiotic, that preferentially targets (G-C).(G-C) steps on duplex DNA. We have reported on the solution structure of the ActD-d(A-A-A-G-C-T-T-T) complex (one drug per duplex) based on a combined application of NMR and molecular dynamics calculations. This study established that ActD binds to DNA through intercalation of the phenoxazone chromophore between (G-C).(G-C) steps with the benzenoid and quinonoid-linked cyclic pentapeptide lactone rings spanning two base-pairs in opposite directions in the minor groove of the helix. This research is now extended to the binding of two ActD molecules to adjacent complexation sites within a (G-C-G-C).(G-C-G-C) segment in the self-complementary d(A1-A2-G3-C4-G5-C6-T7-T8) duplex. The occupation of the central (C4-G5).(C4-G5) segment between the two intercalation sites by the inwardly pointing cyclic pentapeptide lactone rings from adjacent bound ActD molecules should result in a potential steric clash in the center of the helix. The NMR data and its analysis on the ActD-d(A-A-G-C-G-C-T-T) complex (two drugs per duplex) establish that two ActD molecules intercalate into symmetry-related (G3-C4).(G5-C6) steps with their attached benzenoid and quinonoid cyclic pentapeptide lactone rings positioned in the minor groove and directed towards the center and the ends of the helix, respectively. The solution structure of the complex was solved by using NMR restraints to guide distance geometry-simulated annealing and restrained molecular dynamics calculations including intensity-based refinements. The DNA helix exhibits a pronounced kink and is fully unwound at the central (C4-G5).(C4-G5) step which results in an opening and widening of the minor groove to generate additional space for accommodation of the inwardly pointing benzenoid cyclic pentapeptide lactone rings in the complex. The outwardly and inwardly pointing cyclic pentapeptide lactone rings of symmetry-related ActD molecules retain similar conformations with the largest difference observed for the L-MeVal residues in the complex. The present study defines how structural changes primarily in the DNA associated with the directional bending of the helix towards the major groove and away from the bound drug opens up and widens the minor groove to accommodate two intercalated ActD molecules bound at partially overlapping sites on the DNA.

Anti-Bacterial Agents↗

PubMed1996

Molecular recognition in the FMN-RNA aptamer complex.

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.

Base Sequence↗

PubMed1996

Mechanism of the positive inotropic action of cocaine in the guinea pig atrium.

We studied the mechanism of the positive inotropic action of cocaine in isolated guinea pig atria superfused with Tyrode's solution at 31 degrees C while attached to a force transducer to measure peak tension developed, maximum velocity of development of tension, and time to peak tension. Cocaine 2.9 microM enhanced peak tension developed and velocity of development of tension, and prolonged time to peak tension. The increase in peak tension developed produced by cocaine was not affected by propranolol. On the other hand, the cocaine-induced increase in velocity of development of tension was reduced, but not abolished. In the presence of propranolol and phentolamine combined, the cocaine-induced prolongation of time to peak tension was abolished and the increases of both peak tension developed and velocity of development of tension were significantly smaller than those observed in the absence of the two adrenergic blockers. For all practical purposes, nifedipine completely abolished the increase in peak tension developed induced by cocaine. It is concluded that the positive inotropic effect of cocaine in the guinea pig atrial muscle is predominantly the result of adrenergic-dependent, both alpha- and beta- receptor mediated, as well as adrenergic-independent increases in calcium influx through the L-type calcium channels in the sarcolemma.

Adrenergic alpha-Antagonists↗

PubMed1996

Deep penetration of an alpha-helix into a widened RNA major groove in the HIV-1 rev peptide-RNA aptamer complex.

A combined NMR-molecular dynamics approach has been applied to determine the solution structure of a HIV-1 17-mer rev peptide bound to its 35-mer high affinity RNA aptamer binding site. Complex formation involves adaptive binding with the alpha-helical arginine-rich basic rev peptide targeting a widened RNA major groove centred about adjacent G.A and reversed A.A mismatches. We have also identified a U AU triple in the aptamer complex with the Hoogsteen-paired uracil base sandwiched between two arginine side chains. The intermolecular contacts identified in the aptamer complex readily account for the consequences of peptide and RNA mutations, as well as the results of previous in vitro selection experiments. The details of molecular recognition associated with targeting by rev of its high affinity RNA binding sites open new opportunities for structure-based drug design strategies.

Amino Acid Sequence↗

PubMed1996

Encapsulating an amino acid in a DNA fold.

Here we present the first solution structure of a ligand-DNA aptamer complex. Our NMR-molecular dynamics structural studies of the interaction between argininamide and a DNA stem-loop complex establishes that the hairpin loop DNA binding site undergoes an adaptive conformational transition on complex formation. The tip of the DNA loop folds down towards the stem and sandwiches the bound argininamide between reversed Hoogsteen A.C and Watson-Crick G.C base pairs. The argininamide is encapsulated within the structured DNA loop and is stabilized by an intricate set of intermolecular hydrogen bonds and stacking interactions. The structure of the complex lays out the molecular principles defining both the architecture of the internal cavity and the recognition elements that could contribute to ligand discrimination.

Arginine↗

PubMed1996

Early continuous ST segment monitoring in unstable angina: prognostic value additional to the clinical characteristics and the admission electrocardiogram.

BACKGROUND AND OBJECTIVE: In unstable angina, clinical characteristics, resting electrocardiography, and early continuous ST segment monitoring have been individually reported to identify subgroups at increased risk of adverse outcome. It is not known, however, whether continuous ST monitoring provides additional prognostic information in such a setting. DESIGN: Observational study of 212 patients with unstable angina without evidence of acute myocardial infarction admitted to district general hospitals, who had participated in a randomised study comparing heparin and aspirin treatment versus aspirin alone. METHODS: Clinical variables and a 12 lead electrocardiogram (ECG) were recorded at admission, and treatment was standardised to include aspirin, atenolol, diltiazem, and intravenous glyceryl trinitrate, in addition to intravenous heparin (randomised treatment). Continuous ST segment monitoring was performed for 48 h and all inhospital adverse events were recorded. RESULTS: The admission ECG was normal in 61 patients (29%), showed ST depression in 59 (28%) (17 > or = 0.1 mV), and T wave changes in a further 69 (33%). The remaining 23 had Q waves (18), right bundle branch block (four), or ST elevation (one). During 8963 h of continuous ST segment monitoring (mean 42.3 h/patient), 132 episodes of transient myocardial ischaemia (104 silent) were recorded in 32 patients (15%). Forty patients (19%) had an adverse event (cardiac deaths (n = 3), non-fatal myocardial infarction (n = 6) and, emergency revascularisation (n = 31)). Both admission ECG ST depression (P = 0.02), and transient ischaemia (P < 0.001) predicted an increased risk of non-fatal myocardial infarction or death, while no patients with a normal ECG died or had a myocardial infarction. Adverse outcome was predicted by admission ECG ST depression (regardless of severity) (odds ratio (OR) 3.41) (P < 0.001), and maintenance beta blocker treatment (OR 2.95) (P < 0.01). A normal ECG predicted a favourable outcome (OR 0.38) (P = 0.04), while T wave or other ECG changes were not predictive of outcome. Transient ischaemia was the strongest predictor of adverse prognosis (OR 4.61) (P < 0.001), retaining independent predictive value in multivariate analysis (OR 2.94) (P = 0.03), as did maintenance beta blocker treatment (OR 2.85) (P = 0.01) and admission ECG ST depression, which showed a trend towards independent predictive value (OR 2.11) (P = 0.076). CONCLUSIONS: Patients with unstable angina and a normal admission ECG have a good prognosis, while ST segment depression predicts an adverse outcome. Transient myocardial ischaemia detected by continuous ST segment monitoring in such patients receiving optimal medical treatment provides prognostic information additional to that gleaned from the clinical characteristics or the admission ECG.

Adrenergic beta-Antagonists↗

PubMed1996

Natural variability of transient myocardial ischaemia during daily life: an obstacle when assessing efficacy of anti-ischaemic agents?

OBJECTIVE: To assess the degree of variability of transient myocardial ischaemia during daily life in patients with coronary artery disease, which could confound the interpretation of trials of the therapeutic effects of anti-ischaemic agents. DESIGN: Prospective method evaluation. SETTING: Tertiary referral centre, outpatient clinic. PATIENTS: Patients with stable angina, confirmed coronary artery disease, and a positive treadmill exercise test for ischaemia. Patients were not preselected on the basis of prior documented transient ischaemia during ambulatory ST segment monitoring. INTERVENTIONS: A simulated drug-study with 4 monitoring phases in 16 subjects. To minimise variability in ischaemic activity, patients underwent weekly 48 hour ambulatory ST segment monitoring outside hospital off all prophylactic therapy on the same weekdays for 4 weeks. MAIN OUTCOME MEASURE: Variability in the frequency and duration of transient myocardial ischaemia. RESULTS: There was marked variability in both ischaemic activity and mean duration of ischaemia in patients with confirmed ischaemia, the greatest degree of variability being between patients and from day to day within weeks within patients, with a further contribution to variability being noted between fortnights within patients. CONCLUSIONS: Despite assessment off all therapy and an adequate period of monitoring (48 hours) with small intervals between monitoring periods (5 days), marked variability in ischaemic activity was noted, and regression towards the mean was clearly shown. Ambulatory ST segment monitoring outside hospital is not a reliable method for assessing the therapeutic effects of anti-ischaemic agents.

Activities of Daily Living↗

PubMed1995

Solution conformation of [AF]dG opposite a -2 deletion site in a DNA duplex: intercalation of the covalently attached aminofluorene ring into the helix with base displacement of the C8-modified syn guanine and adjacent unpaired 3'-adenine into the major groove.

This paper reports the solution conformation of the covalent aminofluorene-C8-deoxyguanosine [AF]dG adduct positioned opposite a -2 deletion site in a DNA oligomer duplex. The combined NMR and molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-A7-C8).d(G17-G18) sequence context in a duplex containing 12 residues on the modified strand and 10 on the partner strand, with no bases opposite the [AF]dG6-dA7 segment. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]dG.2del 12-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminofluorene ring of [AF]dG6 is intercalated between intact Waston-Crick dC5.dG18 and dC8.dG17 base pairs with the deoxyguanosine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue. The unpaired dA7 base is also looped out of the helix into the major groove with the purine rings of [AF]dG6 and dA7 stacking on each other in the groove. The long axis of the intercalated aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG18 base pair. The deoxyguanosine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative base displacement structure of the [AF]dG.2del 12-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the deoxyguanosine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. We note similarities between the present conformation of [AF]dG positioned opposite a -2 deletion site with our earlier conformational studies of [AF]dG positioned opposite a -1 deletion site [Mao, B., Cosman, M., Hingerty, B. E., Broyde, S., & Patel, D. J. (1995) Biochemistry 34, 6226-6238]. For both conformations, the aminofluorene carcinogen inserts into the helix at the deletion site through base displacement of the modified deoxyguanosine in a syn alignment into the major groove and directed toward its 5'-neighbor in the sequence. These structures provide a molecular explanation of how transient strand slippage of the lesion-containing segment can be accommodated by a double helix following translesion synthesis.

Base Sequence↗

PubMed1995

Solution structure of a DNA quadruplex containing the fragile X syndrome triplet repeat.

Both X-ray and NMR structural studies have defined the polymorphic nature of G-quadruplexes generated through mutual stacking of G.G.G.G tetrads by guanine rich telomeric sequences. Recently, the fragile X syndrome d(C-G-G)n triplet nucleotide repeat has been shown to form a stable quadruplex of undefined structure in monovalent cation solution. We have undertaken a structural characterization of the d(G-C-G-G-T3-G-C-G-G) undecanucleotide to elucidate the structural alignments associated with quadruplex formation by this oligomer which contains sequence elements associated with the fragile X syndrome triplet repeat. d(G-C-G-G-T3-G-C-G-G) in Na+ cation solution forms a quadruplex through dimerization of two symmetry related hairpins with the lateral connecting T3 loops positioned at opposite ends of the quadruplex. This novel NMR-molecular dynamics based solution structure contains internal G.C.G.C tetrads sandwiched between terminal G.G.G.G tetrads. Watson-Crick G.C base-pairs within individual hairpins dimerize through their major groove edges using bifurcated hydrogen bonds to form internal G(anti).C(anti).G(anti).C(anti) tetrads. Adjacent strands are anti-parallel to each other around the symmetric G-quadruplex which contains two distinct narrow and two symmetric wide grooves. By contrast, the terminal G-tetrads adopt G(syn).G(anti).G(syn).G(anti) alignments. The structure of the d(G-C-G-G-T3-G-C-G-G) quadruplex with its multi-layer arrangement of G.G.G.G and G.C.G.C tetrads greatly expands on our current knowledge of quadruplex folding topologies. Our results establish the pairing alignments that can be potentially utilized by the fragile X syndrome triplet repeat to form quadruplex structures through dimerization of hairpin stems. The formation of novel G.C.G.C tetrads through dimerization of Watson-Crick G.C base-pairs is directly relevant to the potential pairing alignments of helical stems in genetic recombination.

DNA↗

PubMed1995

Structural alignments of (+)- and (-)-trans-anti-benzo[a]pyrene-dG adducts positioned at a DNA template-primer junction.

The structural features of a chemically modified DNA template strand may promote error-prone DNA synthesis during replication. The resulting higher incidence of mutations, in turn, can eventually lead to tumor initiation. Structural insights into this process can be monitored by studying chemically modified base adducts of defined stereochemistry positioned site-specifically at a single strand--duplex template--primer junction. We have used a NMR-molecular mechanics approach to obtain the solution conformations of the covalent adducts derived from trans additions at the [BP]C10 position of the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide [(+)-anti-BPDE] and nontumorigenic (-)-anti-benzo-[a]pyrene diol epoxide [(-)-anti-BPDE] to the N2 position of guanine [(+) and (-)-trans-anti-[BP]dG, respectively] in the d(A1-A2-C3-[BP]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13).d (G14-G15-A16-T17-G18-G19-T20-A 21-G22) 13/9-mer DNA sequence. The modified 13-mer strand constitutes the template strand, while the complementary 9-mer strand constitutes a primer which has been synthesized from the 3'-end of the template toward the 5'-end up to the base preceding, but not including, the modified guanine. The modified guanine (denoted by [BP]dG4) is positioned at the junction site between the single-stranded and duplex segments. Structural features of the (+)-trans-anti-[BP]dG 13/9-mer have been determined by incorporating proton--proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The 3'-side duplex segment retains a minimally perturbed B-DNA conformation with all nine base pairs in Watson--Crick hydrogen-bonded alignments. Conformational heterogeneity is detected at the single-stranded d(A1-A2-C3) segment located 5' to the modified (+)-trans-anti-[BP]dG lesion which contrasts with an unperturbed alignment of these same residues in the unmodified control 13/9-mer. The modified guanine adopts a syn glycosidic torsion angle, is displaced into the major groove, and no longer stacks over the adjacent dC5.dG22 base pair. Such a base displacement is accompanied by stacking of one face of the pyrenyl ring with the dC5.dG22 base pair located on the duplex segment proximate to the modified guanine, while the other face of BP is exposed to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed1995

Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

Aminoglycosides↗

PubMed1995

Determination of the folding topology of the SL1 RNA from Caenorhabditis elegans by multidimensional heteronuclear NMR.

The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In this study, the first stem loop of the SL1 RNA from the nematode Caenorhabditis elegans was examined by homonuclear and heteronuclear NMR. Results of enzymatic cleavage patterns established that the first 36 nucleotides (which includes the splice site and a complementary base-paired region surrounding a nine-nucleotide hairpin loop) remain structurally independent of the rest of the 100-nucleotide full-length transcript. A comparison of exchangeable and non-exchangeable proton chemical shifts in the region of the splice site and loop between the native sequence and a modified 26-nucleotide fragment from which an asymmetric internal loop had been deleted was made. There was no significant difference between the resonance locations of the equivalent protons in the two molecules, establishing that there was no tertiary interaction between the hairpin and internal loops. Full chemical shift assignments of 1H, 13C, and 15N chemical shifts were obtained for the modified fragment by multidimensional homonuclear and heteronuclear NMR spectroscopy. The stem adopts an A-form helix typical of RNA. The A-type helical conformation of the stem appears to continue for the first three nucleotides of the 5' side of the loop, followed by a guanosine residue in a syn conformation about the glycosidic bond. Base stacking is not seen on the 3' side of the loop. There was no evidence for formation of Watson-Crick base-pairs within the loop, but several long distance NOEs indicated cross-loop contacts, indicative of a structured loop. The final loop residues, an adenine which is conserved among all known nematode SL RNA sequences, adopts an extrahelical conformation.

Animals↗

PubMed1995

Solution structure of mithramycin dimers bound to partially overlapping sites on DNA.

Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg(2+)-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C).(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C).(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA 10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A).(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.

Base Composition↗

PubMed1995

Solution structure of the Oxytricha telomeric repeat d[G4(T4G4)3] G-tetraplex.

The solution structure of Oxytricha telomere sequence d[G4(T4G4)3] in 0.1 M Na+ containing solution has been determined using a combined NMR-molecular dynamics approach including relaxation matrix refinement. This four G4 repeat sequence folds intramolecularly into a right-handed G-tetraplex containing four stacked G-tetrads which are connected by two lateral T4 loops and a central diagonal T4 loop. The guanine glycosidic bonds adopt a syn-anti alternation along the full length of the d[G4(T4G4)3] sequence while the orientation around adjacent G-tetrads switches between syn.syn.anti.anti and anti.anti.syn.syn alignments. Four distinct grooves are formed by the parallel (two of medium width) and anti-parallel (one wide and one narrow width) alignment of adjacent G-G-G-G segments in the G-tetraplex. The T4 residues in the diagonal loop are well-defined while the T4 residues in both lateral loops are under-defined and sample multiple conformations. The solution structure of the Na(+)-stabilized Oxytricha d[G4(T4G4)3] G-tetraplex and an earlier solution structure reported from our laboratory on the Na(+)-stabilized human d[AG3(T2AG3)3] G-tetraplex exhibit a common folding topology defined by the same syn/anti distribution of guanine residues along individual strands and around individual G-tetrads, as well as a common central diagonal loop which defines the strand directionalities. The well-resolved proton NMR spectra associated with the d[G4(T4G4)3] G-tetraplex opens the opportunity for studies ranging from cation-dependent characterization of G-tetraplex conformation and hydration to ligand and protein recognition of the distinct grooves associated with this folding topology.

Animals↗