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D J Patel

Publications and source records attributed to D J Patel.

At least 127 records · Page 7Linked to original sources
PubMed1994

Hydration sites in purine.purine.pyrimidine and pyrimidine.purine.pyrimidine DNA triplexes in aqueous solution.

BACKGROUND: DNA triplexes are higher-order nucleic acid structures with potential roles in gene regulation and hence biochemical and therapeutic applications. The stabilizing influence exerted by water molecules on the conformation of the DNA duplex is well known. However, the role of water molecules in the DNA triple helix has not been investigated. We have previously determined the solution structures of the purine.purine.pyrimidine (R.RY) and pyrimidine.purine.pyrimidine (Y.RY) structural motifs in DNA triplexes and identified both the global helical parameters, as well as local helical distortions associated with non-standard base triple pairing alignments. RESULTS: Here we have used homonuclear two-dimensional NMR spectroscopy to define the hydration sites in R.RY and Y.RY DNA triplexes in aqueous solution. Long-lived hydration sites with residence times exceeding 1 nanosecond have been identified in the new groove formed by the Hoogsteen paired strands in both triplexes. Distinctive patterns of hydration are displayed by each triplex in the remaining two grooves. CONCLUSION: The role played by water molecules in DNA triplexes appears to be similar to that played in duplexes. By binding to specific sites, particularly in the narrow groove formed by the Hoogsteen paired strands whose phosphate groups are in close proximity, water molecules may stabilize the triplex by shielding it against unfavorable electrostatic interactions.

Base Composition↗
PubMed1994

Solution structure of a pyrimidine.purine.pyrimidine DNA triplex containing T.AT, C+.GC and G.TA triples.

BACKGROUND: Under certain conditions, homopyrimidine oligonucleotides can bind to complementary homopurine sequences in homopurine-homopyrimidine segments of duplex DNA to form triple helical structures. Besides having biological implications in vivo, this property has been exploited in molecular biology applications. This approach is limited by a lack of knowledge about the recognition by the third strand of pyrimidine residues in Watson-Crick base pairs. RESULTS: We have therefore determined the solution structure of a pyrimidine.purine.pyrimidine (Y.RY) DNA triple helix containing a guanine residue in the third strand which was postulated to specifically recognize a thymine residue in a Watson-Crick TA base pair. The structure was solved by combining NMR-derived restraints with molecular dynamics simulations conducted in the presence of explicit solvent and counter ions. The guanine of the G-TA triple is tilted out of the plane of its target TA base pair towards the 3'-direction, to avoid a steric clash with the thymine methyl group. This allows the guanine amino protons to participate in hydrogen bonds with separate carbonyls, forming one strong bond within the G-TA triple and a weak bond to an adjacent T.AT triple. Dramatic variations in helical twist around the guanine residue lead to a novel stacking interaction. At the global level, the Y.RY DNA triplex shares several structural features with the recently solved solution structure of the R.RY DNA triplex. CONCLUSIONS: The formation of a G.TA triple within an otherwise pyrimidine.purine.pyrimidine DNA triplex causes conformational realignments in and around the G.TA triple. These highlight new aspects of molecular recognition that could be useful in triplex-based approaches to inhibition of gene expression and site-specific cleavage of genomic DNA.

Base Composition↗
PubMed1993

Solution structure of a parallel-stranded G-quadruplex DNA.

This paper reports on the solution structure of a parallel-stranded G-quadruplex formed by the Tetrahymena telomeric sequence d(T-T-G-G-G-G-T) whose NMR parameters in potassium cation containing solution were previously published from our laboratory. The structure was determined by combining a quantitative analysis of the NMR data with molecular dynamics calculations including relaxation matrix refinement. The combined NMR-computational approach yielded a set of seven distance-refined structures with pairwise RMSDs ranging from 0.66 to 1.30 A for the central G-G-G-G tetranucleotide segment. Four of the seven structures were refined further using complete relaxation-matrix calculations to yield solution structures with pairwise RMSDs ranging from 0.64 to 1.04 A for the same tetranucleotide segment. The R-factors also decreased on proceeding from the distance-refined to relaxation matrix-refined structures. The four strands of the G-quadruplex are aligned in parallel and are related by a 4-fold symmetry axis coincident with the helix axis. Individual guanines from each strand form planar G.G.G.G tetrad arrangements with each tetrad stabilized by eight hydrogen bonds involving the Watson-Crick and Hoogsteen edges of the guanine bases. All guanines adopt anti glycosidic torsion angles and S type sugar puckers in this right-handed parallel-stranded G-quadruplex structure. The four G.G.G.G tetrad planes stack on each other with minimal overlap of adjacent guanine base planes within individual strands. The thymine residues are under-defined in the solution structure of the d(T-T-G-G-G-G-T) G-quadruplex and sample amongst multiple conformations in solution.

DNA↗
PubMed1993

Solution structure of the human telomeric repeat d[AG3(T2AG3)3] G-tetraplex.

BACKGROUND: Repeats of Gn sequences are detected as single strand overhangs at the ends of eukaryotic chromosomes together with associated binding proteins. Such telomere sequences have been implicated in the replication and maintenance of chromosomal termini. They may also mediate chromosomal organization and association during meiosis and mitosis. RESULTS: We have determined the three-dimensional solution structure of the human telomere sequence, d[AG3(T2AG3)3] in Na(+)-containing solution using a combined NMR, distance geometry and molecular dynamics approach (including relaxation matrix refinement). The sequence, which contains four AG3 repeats, folds intramolecularly into a G-tetraplex stabilized by three stacked G-tetrads which are connected by two lateral loops and a central diagonal loop. Of the four grooves that are formed, one is wide, two are of medium width and one is narrow. The alignment of adjacent G-G-G segments in parallel generates the two grooves of medium width whilst the antiparallel arrangement results in one wide and one narrow groove. Three of the four adenines stack on top of adjacent G-tetrads while the majority of the thymines sample multiple conformations. CONCLUSIONS: The availability of the d[AG3(T2AG3)3] solution structure containing four AG3 human telomeric repeats should permit the rational design of ligands that recognize and bind with specificity and affinity to the individual grooves of the G-tetraplex, as well as to either end containing the diagonal and lateral loops. Such ligands could modulate the equilibrium between folded G-tetraplex structures and their unfolded extended counterparts.

Base Sequence↗
PubMed1993

Nuclear magnetic resonance structural studies of A.AT base triple alignments in intramolecular purine.purine.pyrimidine DNA triplexes in solution.

This study reports on an NMR characterization of an intramolecular purine.purine.pyrimidine (R.RY) triplex containing a central A.AT triple in addition to G.GC and T.AT triples. Our studies establish an A(anti).AT alignment with reversed Hoogsteen pairing stabilized by two N6H ... N hydrogen bonds between adenine residues in the third strand and the purine strand of the duplex. This result, combined with our earlier demonstration of G(anti).GC and T(anti).AT pairing alignments, has provided a definitive experimental approach for differentiating between the base triple pairing possibilities proposed previously by Beal and Dervan for R.RY triplexes.

DNA↗
PubMed1993

NMR structural studies on a nonnatural deoxyribonucleoside which mediates recognition of GC base pairs in pyrimidine-purine-pyrimidine DNA triplexes.

As a part of our ongoing efforts to define the structural aspects of unusual pairing alignments in DNA triplexes by nuclear magnetic resonance spectroscopy, we have examined the structural role of a nonnatural deoxyribonucleoside, P1, that has been shown to mediate the recognition of GC base pairs in pyrimidine-purine-pyrimidine DNA triplexes [Koh, J.S., & Dervan, P.B. (1992) J. Am. Chem Soc. 114, 1470]. A qualitative interpretation of the NMR data indicates that this analog of protonated cytosine is readily accommodated in the third strand segment of an intramolecular triplex system. Furthermore, the observed NOE patterns position the imino and amino protons of P1 opposite the N7 and O6 atoms of guanine, respectively, consistent with the previously proposed pairing scheme.

Base Composition↗
PubMed1993

Solution structure of a purine.purine.pyrimidine DNA triplex containing G.GC and T.AT triples.

BACKGROUND: Oligonucleotide-directed triple helix formation allows sequence specific recognition of double helical DNA. This powerful approach has been used to inhibit gene transcription in vitro and to mediate single site specific cleavage of a human chromosome. RESULTS: Using a combined NMR and molecular dynamics approach (including relaxation matrix refinement), we have determined the solution structure of an intramolecular purine.purine.pyrimidine (R.RY) DNA triplex containing guanines and thymines in the third strand to high resolution. Our studies define the G.GC and T.AT base triple pairing alignments in the R.RY triplex and identify the structural discontinuities in the third strand associated with the non-isomorphism of the base triples. The 5'-d(TpG)-3' base steps exhibit a pronounced increase in axial rise and reduction in helical twist, while the reverse is observed, to a lesser extent at 5'-d(GpT)-3' steps. A third groove is formed between the purine-rich third strand and the pyrimidine strand. It is wider and deeper than the other two grooves. CONCLUSIONS: Our structure of the R.RY DNA triplex will be important in the design of oligonucleotide probes with enhanced specificity and affinity for targeting in the genome. The third groove presents a potential target for binding additional ligands.

Adenine↗
PubMed1993

Conformational differences between bulged pyrimidines (C-C) and purines (A-A, I-I) at the branch point of three-stranded DNA junctions.

We have synthesized DNA oligomers that can combine to form three-way junctions containing six base pairs in each stem and two unpaired bases at the branch point. Gel electrophoresis experiments indicate that the oligomers form stable complexes with equimolar stoichiometry. Using two- and three-dimensional proton nuclear magnetic resonance spectroscopy, we have completed nonexchangeable proton chemical shift assignments for three junctions which differ only in the identity of the unpaired bases (C-C, A-A, or I-I) at the branch point. Our results indicate that unpaired pyrimidines at the branch point of junctions behave differently than do unpaired purines. In a junction with two unpaired cytidines, the 5' base loops out from the molecule to lie along the minor groove of the preceding duplex stem of the junction. The 3' unpaired cytidine also demonstrates an unusual pattern of NOE connectivities with detected cross peaks to the subsequent base in the 3' direction. Junctions with unpaired purines at the branch point exhibit different behavior. Our data suggests that in these molecules the unpaired bases participate in stacking interactions among themselves and with the neighboring bases in the molecule. Despite these differences, the NOE patterns from each junction suggest the presence of a preferred, pair-wise stacking between two of the helices within the molecule. The structural differences between bulge-pyrimidine and bulge-purine junctions are discussed in light of the functional significance unpaired bases might have in the structure and dynamics of multistranded DNA junctions and, by extension, to junctions within cellular RNAs.

Adenosine↗
PubMed1993

Structural features of a three-stranded DNA junction containing a C-C junctional bulge.

We have examined the stability of junctional base pairs in a three-way DNA junction with two unpaired cytidine residues at the branch point using two-dimensional nuclear Overhauser effect spectroscopy in H2O solution. Our data directly support the presence of two of the three junctional Watson-Crick base pairs, with indirect support for the third as well. These results complement the data presented in the preceding paper, where we examined the nonexchangeable proton resonance assignments of three-way DNA junctions from NOESY data in D2O solution. We have incorporated the NOE data from both sets of experiments, using this information as input for a combined distance geometry (DG) and simulated annealing (SA) protocol designed to derive three-dimensional structures of the junction molecule consistent with the NMR data. Although the data does not allow us to derive a unique solution for the structure of the molecule, certain conformational features are invariably present in our models. We demonstrate the existence of a preferred, pair-wise stacking arrangement between two of the three helices in the junction. Furthermore, the remaining duplex stem is situated so that it always forms an acute angle with just one of the arms from the quasi-continuous helix. The unpaired residues provide an extended backbone segment linking two of the helices together. The first unpaired base on the 5' end loops out from the interior of the molecule to reside along the minor groove of one helix. The second is located within the interior of the molecule, stacking below one of the junctional base pairs. Our findings suggest that junctional base pair stacking is an important determinant in the conformation of multistranded nucleic acid junctions. In three-way junctions, the presence of unpaired bases at the branch point provides a relief from covalent constraints that would otherwise prevent the simultaneous realization of both base pairing and base pair stacking within the branch point of the molecule.

Base Composition↗
PubMed1993

Solution structure of the mithramycin dimer-DNA complex.

We have characterized the NMR parameters for the complexes formed by the Mg(2+)-coordinated mithramycin dimer with self-complementary d(T-G-G-C-C-A) and d(T-C-G-C-G-A) duplexes. The solution structure of the latter complex has been determined using a combined NMR-molecular dynamics study including relaxation matrix refinement. The Mg(2+)-coordinated mithramycin dimer-d(T-C-G-C-G-A) complex exhibits a 2-fold center of symmetry with the divalent cation coordinated aglycons positioned opposite the central (G3-C4).(G3-C4) segment such that the aglycon C8 hydroxyl oxygens form symmetrical sequence-specific hydrogen bonds to guanine amino protons in the complex. The C-D-E trisaccharide segments of each monomer in the mithramycin dimer adopt extended conformations, are positioned inside the minor groove, and are directed toward either end of the duplex. The C-D saccharide component of one monomer and the aglycon of the other monomer in the mithramycin dimer share a widened minor groove with the hydrophobic edges of the C and D sugars interacting with individual strands of the duplex. The E-sugar ring is positioned in the floor of the minor groove, and its hydroxyl-bearing face interacts with both strands of the duplex through hydrogen-bonding and hydrophobic intermolecular interactions. The A-B disaccharide and the hydrophilic side chain form intermolecular contacts with the sugar-phosphate backbone in the complex. The antiparallel alignment of divalent cation coordinated monomers in the mithramycin dimer results in the two outwardly directed C-D-E trisaccharide segments generating a right-handed continuous hexasaccharide domain that spans six base pairs in the minor groove of the duplex. The solution structure of the mithramycin dimer-DNA complex reported in this study and the solution structure of the chromomycin dimer-DNA complex reported previously [Gao, X., Mirau, P., & Patel, D. J. (1992) J. Mol. Biol. 223, 259-279] show global similarities, as well as local differences that are of interest. All four nucleotides in the tetranucleotide segment of the duplex centered about the sequence-specific (G-C).(G-C) step adopt A-DNA sugar puckers and glycosidic torsion angles in the chromomycin dimer-DNA complex, while only the central cytidine adopts an A-DNA sugar pucker and glycosidic torsion angle in the mithramycin dimer-DNA complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1993

Solution conformation of the (+)-cis-anti-[BP]dG adduct in a DNA duplex: intercalation of the covalently attached benzo[a]pyrenyl ring into the helix and displacement of the modified deoxyguanosine.

This paper reports on the solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite dC in a DNA oligomer duplex which provides the first experimentally based solution structure of an intercalative complex of a polycyclic aromatic hydrocarbon covalently bound to the N2 of deoxyguanosine. The combined NMR-energy minimization computation studies were undertaken on the (+)-cis-anti-[BP]dG adduct embedded in the same d(C5-[BP]G6-C7).d(G16-C17-G18) trinucleotide segment of the complementary 11-mer duplex studied previously with the stereoisomeric trans adducts. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY data sets as restraints in energy minimization computations. The benzo[a]pyrene ring of [BP]dG6 is intercalated between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs in a right-handed DNA helix. The benzylic ring is in the minor groove while the pyrenyl ring sacks with flanking dC5 and dC7 bases on the same strand. The deoxyguanosine ring of [BP]dG6 is not Watson-Crick base paired but displaced into the minor groove with its plane parallel to the helix axis and stacks over the sugar ring of dC5. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove by the intercalated benzo[a]pyrene ring. This intercalative structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex exhibits several unusually shifted proton resonances which can be readily accounted for by the ring current contributions of the deoxyguanosine and pyrenyl rings of the [BP]dG6 adduct. Several phosphorus resonances are shifted to low and high field of the unperturbed phosphorus spectral region and have been assigned to internucleotide phosphates centered about the [BP]dG6 modification site. These studies define the changes in the helix at the central trinucleotide segment needed to generate the intercalation site for the covalently bound (+)-cis-anti-[BP]dG adduct.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Solution structure of the exocyclic 1,N2-propanodeoxyguanosine adduct opposite deoxyadenosine in a DNA nonamer duplex at pH 8.9. model of pH-dependent conformational transition.

The solution structure of the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A16-T17-G18) DNA duplex (designated X.A 9-mer), which contains a 1,N2-propanodeoxyguanosine exocyclic adduct X5 opposite deoxyadenosine A14 at the center, is pH dependent [Kouchakdjian, M., Eisenberg, M., Live, D., Marinelli, E., Grollman, A., & Patel, D.J. (1990) Biochemistry 29, 4456-4465]. In our previous paper [Huang, P., & Eisenberg, M. (1992) Biochemistry 31, 6518-6532] we established the three-dimensional structure of this X.A 9-mer duplex at pH 5.8 by use of restrained molecular dynamics followed by NOE-based back-calculation refinement. The present paper discusses the structure at pH 8.9 and the pH-dependent conformational transition between the structures at pH 5.8 and at pH 8.9. The structure at pH 8.9 is calculated starting from five different conformations. The final structures converge and agree well with the experimental NOE intensities. These structures are essentially B-type DNA (with X5 and A14 in the BII conformation while the other residues are in the most commonly described BI conformation) and display an approximate 27 degrees kink at the center of the helix. At the kink site, X5 is positioned in the major groove with the exocyclic ring directed toward the G6.C13 base pair, unstacked from the flanking base G6 and exposed to the solvent. A14, opposite the lesion, remains stacked with its neighbor C15, but not with C13. The kinked helix can accommodate the rotation of the bulky X5 about its glycosidic bond. We propose here a model for the pH-dependent transition. Our model explains the conformational change, which includes the anti and syn rotation of the bulky adduct around its glycosidic bond, with a minimal energy barrier and with an overall kink of the DNA helix. These new findings, fully consistent with the NMR experimental data, were revealed only after restrained dynamics refinement. Distance-restrained energy minimization by itself was insufficient, as shown by the previous NMR study.

Base Sequence↗
PubMed1993

The antitumor drug nogalamycin forms two different intercalation complexes with d(GCGT).d(ACGC).

The structures of the physical complex of d(GCGT).d(ACGC) with the anthracycline antitumor drug nogalamycin were studied in order to determine the sequence specificity and the drug orientation at the symmetric d(C2G3).d(C6G7) binding site of this oligonucleotide. For this purpose, one- and two-dimensional NMR techniques were used in combination with molecular mechanics and molecular dynamics computations. Analysis of the NMR spectra reveals that nogalamycin forms two different intercalation complexes with d(GCGT).d(ACGC). These complexes are called complex I and complex II and are present in a ratio of 0.45:0.55. In both complexes the nogalamycin is intercalated at the d(C2G3).d(C6G7) sequence with the bicyclic and nogalose sugars residing in the major and minor groove, respectively. This results in a buckling of the flanking base pairs and a doubling of the inter-base-pair distances at the intercalation site. In complex I, the aglycon ring of the drug stacks with the C6-G7 bases, and the sugars are directed to the G1.C8 end; while in the case of complex II the anthraquinone ring system is stacked with C2-G3 bases, and the sugars are pointed to the T4.A5 base pair end. The two nogalamycin-d(GCGT).d(ACGC) structures are stabilized by intra- and intermolecular hydrogen bonds, electrostatic interactions, and van der Waals contacts. Comparison of different nogalamycin-oligonucleotide structures reveals a nogalamycin binding specificity to the 3'-side of the cytosine base in cytosine-purine sequences in double-stranded DNA.

Base Composition↗
PubMed1993

Mechanism of the negative inotropic action of cocaine in rat papillary muscle.

We studied the mechanism of the negative inotropic action of cocaine in isolated rat papillary muscles superfused with Tyrode's solution at 31 degrees C while attached to a force transducer to measure peak tension developed (PTD), maximum velocity of development of tension (Vmax T), and time to peak tension (TPT). The results show that in preparations driven at 1 Hz, 5 and 10 mg/liter cocaine had a significant negative effect only on PTD, while 20 mg/liter (58 microM) significantly depressed all three measured parameters. Similar results were obtained in preparations driven at 0.5 Hz, which developed a greater control active tension than those driven at 1 Hz. In preparations driven at 0.5 Hz, propranolol (1 microM) had no significant effect on the active tension developed nor did it modify the negative inotropic action of cocaine. Both nifedipine (1 microM) and caffeine (1.5 mM), on the other hand, significantly reduced PTD, Vmax T, and TPT. The depressant inotropic action of cocaine was accentuated by nifedipine, but was blocked by caffeine. These observations suggest that the primary site of action of cocaine was at the level of the sarcoplasmic reticulum rather than at the sarcolemma.

Animals↗
PubMed1993

Distensibility of small pulmonary blood vessels.

Although vasomotor activity in small pulmonary vessels has been studied extensively in the past, using the concept of resistance to flow, information on the distensibility of these vessels is very sparse. In an attempt to reduce this deficit, we adapted a theoretical method developed for small systemic vessels, to estimate distensibility of pulmonary resistance vessels in experimental animals and man. Pressure-flow data from 11 dogs and 10 human subjects (5 control subjects and 5 patients with long-standing left heart failure) were used to calculate distensibility of small pulmonary vessels. The conductance, G, was calculated from these data as the ratio of blood flow to driving pressure. The slope of the relationship between the logarithm of G1/4 and the average distending pressure (ADP) provides a graphic picture of circumferential extensibility, E, defined as percent change in radius for an infinitesimal change in ADP. Results indicate that: (1) the value of E in dogs was 1.85 +/- .40 mmHg-1 for the control state, which decreased to 1.45 +/- .43 mmHg-1 during norepinephrine administration; however, the decrease in the value was not statistically significant (p = 0.53); (2) the value of E in control human subjects was 3.38 +/- .47 mmHg-1 and the value of E in patients with left heart failure was -0.64 +/- 0.39 mmHg-1; the difference was significant (P = .0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗