Search PubMed⌕ Search

Biomedical subjects

G Zon

Publications and source records attributed to G Zon.

At least 73 records · Page 4Linked to original sources
PubMed1992

Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells.

A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for cellular uptake and subcellular distribution. The relationship between extracellular oligonucleotide concentration and the distribution and accumulation into subcellular organelles is important to the design, potential side effects, and understanding of a therapeutically useful antisense oligonucleotide. These studies employed uptake of both 35S- and fluorescence-labeled phosphorothioate oligonucleotides. Experiments with V79, HeLa, H9, and fresh human peripheral blood monocytes indicate that accumulations of oligonucleotide inside cells exceeds the concentration of oligonucleotide in culture media by over 100 times following 1 h of exposure at 37 degrees C. Uptake is more efficient at low concentrations, suggesting a saturable process. The total oligonucleotide that remains in cells begins to reach a plateau after 45-60 min, indicating either that efflux pathways exist or that uptake is saturable. Subcellular fractionation studies with 35S-labeled phosphorothioate demonstrate the oligonucleotide is sequestered into both the nuclei and the mitochondria of cultured HeLa cells in a time-dependent manner. The subcellular fractionation was examined with fluorescence-labeled phosphorothioate by both confocal and fluorescence microscopy, which confirmed the rate and localization of oligonucleotide into cultured cells. Finally, cellular uptake is not uniform for all cells in a nonsynchronous culture.

Animals↗
PubMed1992

Binding of antisense phosphorothioate oligonucleotides to murine lymphocytes is lineage specific and inducible.

A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for its ability to associate with murine lymphoid cells. The relationship between cellular oligonucleotide concentration and the lymphoid target tissues is important to the selection of an animal model, evaluation of potential side effects, and understanding the actions of a therapeutically useful antisense oligonucleotide. Lymphoid cells were harvested from murine peripheral blood, bone marrow, thymus, lymph node, and spleen. Cell subpopulations that bind the oligonucleotide were distinguished by two-color flow cytometry employing a fluorescein-labeled anti-rev oligonucleotide and phycoerythrin-labeled antibodies to selected cell surface molecules associated with unique subpopulations of cells. Very little oligonucleotide binding was observed in peripheral blood mononuclear cells or thymic T cells, but substantial numbers of cells, primarily B cells from bone marrow and spleen, accumulated the oligonucleotide. The cell-associated oligonucleotide was increased significantly in lymphoid populations when the cells were mitogen pretreated with either concanavalin-A (ConA), a T cell mitogen, or lipopolysaccharide (LPS), a B cell mitogen. These data clearly demonstrate the ability of fluorescein-conjugated oligonucleotides to bind to unique cell populations in suspension, allowing simultaneous two-color phenotypic analysis, suggesting that fluorescein-conjugated oligonucleotides may be a useful bridge between in vitro molecular biology techniques and in vivo cell biology. In addition, these data provide optimism concerning the in vivo treatment of chronically infected HIV patients using antisense oligonucleotides.

Animals↗
PubMed1992

Oligodeoxyribonucleotide phosphorothioate fluxes and localization in hematopoietic cells.

An antisense oligonucleotide phosphorothioate, previously shown to inhibit HIV-1 viral expression in chronically infected H9 cells, was fluorescently labeled to study oligonucleotide fluxes and localization within living cells. Observations based on flow cytometry and fluorescence microscopy show the following: within around 0.5-2 h, an apparent steady-state distribution of the oligonucleotide is achieved in which the intracellular oligonucleotide concentration is less than that present in the external medium; following oligonucleotide uptake and resuspension of the cells in oligonucleotide-free medium, an oligonucleotide efflux, with a time constant similar to that for uptake, is observed (although a significant fraction of the phosphorothioate remains within the cell); cellular uptake as a function of the external oligonucleotide concentration is nonlinear, being more efficient at lower concentrations (less than 2 microM); and a predominant oligonucleotide localization within the cell nucleus and perinuclear organelles is observed.

Base Sequence↗
PubMed1992

Antisense oligonucleotides.

Growing evidence indicates that antisense oligodeoxynucleotides can specifically inhibit gene expression thereby providing an essential tool for understanding gene function and the potential to affect abnormal cell proliferation. Because oncogene activation is intimately involved in tumour initiation and progression, down-regulation of oncogene expression is associated with a selective or a preferential inhibition of tumour as compared to normal cell proliferation. Even though numerous studies attest the short-term in vitro efficacy of antisense oligodeoxynucleotides as inhibitors of tumour growth, the use of these compounds as therapeutic agents awaits a more rigorous demonstration of their long term effects and favourable pharmacological properties.

Antineoplastic Agents↗
PubMed1991

Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1991

Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers.

The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations.

Base Sequence↗
PubMed1991

Structure determination of [d(ATATATAUAT)]2 via two-dimensional NOE spectroscopy and molecular dynamics calculations.

Proton homonuclear two-dimensional (2D) NOE spectra were obtained for the decamer [d(ATATATAUAT)]2 as a function of mixing time, and proton resonance assignments were made. Quantitative assessment of the 2D NOE cross-peak intensities was used in conjunction with the program MARDIGRAS, which entails a complete relaxation matrix analysis of the 2D NOE peak intensities, to obtain a set of upper and lower bound interproton distance constraints. The analysis with MARDIGRAS was carried out using three initial models: A-DNA, B-DNA and Z-DNA. The distance constraints determined were essentially the same regardless of initial structure. These experimental structural constraints were used with restrained molecular dynamics calculations to determine the solution structure of the decamer. The molecular dynamics program AMBER was run using A-DNA or B-DNA as starting model. The root-mean-square (rms) difference between these two starting models is 0.504 nm. The two starting models were subjected to 22.5 ps of restrained molecular dynamics calculations. The coordinates of the last 10.5 ps of the molecular dynamics runs were averaged to give two final structures. MDA and MDB. The rms difference between these two structures is 0.09 nm, implying convergence of the two molecular dynamics runs. The 2D NOE spectral intensities calculated for the derived structures are in good agreement with experimental spectra, based on sixth-root residual index analysis of intensities. A detailed examination of the structural features suggests that while the decamer is in the B-family of DNA structures, many torsion angle and helical parameters alternate from purine to pyrimidine, with kinks occurring at the U-A steps.

Base Sequence↗
PubMed1991

Hexagonal crystal structure of the A-DNA octamer d(GTGTACAC) and its comparison with the tetragonal structure: correlated variations in helical parameters.

The alternating DNA octamer d(GTGTACAC) has been grown in a novel hexagonal crystal form. The structure has been determined and refined to a 2-A resolution, with 51 water molecules. The A-DNA conformation is a variant of that observed for the tetragonal form of the same sequence (Jain et al., 1989) containing a bound spermine. The crystals belong to the space group P6(1)22, a = b = 32.40 A and c = 79.25 A, with one strand in the asymmetric unit. The new hexagonal structure was solved by rotation and translation searches in direct space and refined to a final R value of 12.7% by using 1561 unique reflections greater than 1.5 sigma (I). The electron density clearly shows that the penultimate A7 sugar had flipped into the alternative C2'-endo pucker. This dent in the molecule can be attributed to close intermolecular contacts. In contrast, in the tetragonal structure, the DNA is distorted in the central TA step, where the A5 backbone bonds C4'-C5' and O5'-P assume trans conformations. The hexagonal double helix more closely resembles the fiber diffraction A-DNA, compared to the tetragonal form. For instance, the tilt angle is higher (16 degrees vs 10 degrees), which is correlated with a larger displacement from the helix axis (3.5 vs 3.3), a lower rise per residue (2.9 vs 3.2), and a smaller major-groove width (6.1 vs 8.7), thus indicating that the variations in these global helical parameters are correlated. The propeller twist angles in both forms are higher for the G-C base pairs (15.3 degrees, 12.14 degrees) than for the A-T base pairs (10.8 degrees, 9.1 degrees), which is the reverse of the expected order. Unlike the tetragonal structure, the hexagonal crystal structure interestingly does not contain a bound spermine molecule. Our analysis reveals that the conformational differences between the tetragonal and hexagonal forms are not entirely due to the spermine binding, and crystal packing seems to play an important role.

Base Composition↗
PubMed1991

NMR and molecular modeling evidence for a G.A mismatch base pair in a purine-rich DNA duplex.

1H NMR experiments indicate that the oligomer 5'-d(ATGAGCGAATA) forms an unusual 10-base-pair duplex with 4 G.A base pairs (underlined) and a 3' unpaired adenosine. NMR results indicate that guanosine imino protons of the G.A mismatches are not hydrogen bonded but are stacked in the helix. A G----I substitution in either G.A base pair causes a dramatic decrease in duplex stability and indicates that hydrogen bonding of the guanosine amino group is critical. Nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) results indicate that the overall duplex conformation is in the B-family. Cross-strand NOEs in two-dimensional NOESY spectra between a mismatched AH2 and an AH1' of the other mismatched base pair and between a mismatched GH8 and GNH1 of the other mismatch establish a purine-purine stacking pattern, adenosine over adenosine and guanosine over guanosine, which strongly stabilizes the duplex. A computer graphics molecular model of the unusual duplex was constructed with G.A base pairs containing A-NH2 to GN3 and G-NH2 to AN7 hydrogen bonds and B-form base pairs on both sides of the G.A pairs [5'-d(ATGAGC)]. The energy-minimized duplex satisfies all experimental constraints from NOESY and COSY results. A hydrogen bond from G-NH2 of the mismatch to a phosphate oxygen is predicted.

Adenine↗
PubMed1991

Caesium fluoride-induced changes in the c.d. spectra of synthetic DNA fragments.

Ten DNA fragments containing self-complementary alternating sequences of adenine and thymine differing in length and the starting nucleotide were studied by c.d. spectroscopy. It was found that d(TATATATA) but not d(ATATATAT), d(TATATA), d(CTATATAG) or (dT-dA)20 isomerized into the unusual X-DNA double helix at molar concentrations of CsF in solution. But in contrast to poly(dA-dT), the octamer (dT-dA)4, isomerized very slowly, at relatively low CsF concentrations and the isomerization was strongly dependent on the octamer concentration. A model is proposed to account for the observed properties of the B-to-X isomerization on the oligomer level.

Adenine Nucleotides↗
PubMed1991

Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions.

There is widespread and growing interest in the use of phosphorothioate-modified oligonucleotides as sequence-specific agents to control transcription, splicing, translation and other regulated processes in vitro and in vivo, as pharmaceuticals. These exciting applications are predicated on the resistance of phosphorothioate oligonucleotides to degradation by nucleases, a property that results simply from incorporation of sulfur into the phosphate backbone. The popularity of phosphorothioate oligonucleotide analogs derives from several of their features: relatively easy automated synthesis, uncomplicated purification and handling, plus high solubility in water. Presented here are current preparative and analytical methods, together with a comprehensive comparative analysis of solid-phase and solution processes for manufacturing phosphorothioate oligonucleotides as bulk pharmaceutical compounds for clinical evaluation. A prospective view of the future is offered with the hope of directing attention to important areas of needed research dealing with phosphorothioate oligonucleotide technology in particular, and manufacturing of nucleic acid pharmaceuticals in general.

Base Sequence↗
PubMed1990

Two-dimensional COSY and two-dimensional NOE spectroscopy of d(AC)4.d(GT)4: extraction of structural constraints.

Pure absorption phase, proton two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (DQF-COSY) spectra were recorded for d(AC)4.d(GT)4. A full proton resonance assignment was made, except for the 5' and 5" protons. A new semiautomatic method for improved quantitation of 2D NOE peak intensities was developed, and its limitations and usefulness were examined. With this new method, 2D NOE intensity sets at several mixing times were obtained. Simulations of the 1'2', 1'2", and 2'3' DQF-COSY cross-peaks were compared with experimental data, establishing an alternating sugar pucker for the alternating purine-pyrimidine sequence. Scalar coupling constants for the sugar ring protons, derived from the fitting of the simulated spectra, are reported. Complete relaxation matrix analysis of the 2D NOE spectrum verified this alternating structure for all NOE interactions between nonexchangeable protons. Both the DQF-COSY and the 2D NOE results qualitatively indicate that the structure of d(AC)4.d(GT)4 resembles wrinkled D-DNA in aqueous solution.

Computer Simulation↗
PubMed1990

Interaction of a macrocyclic bisacridine with DNA.

The binding of the macrocycle SDM to DNA was investigated by visible spectroscopy, stopped-flow kinetics, and NMR spectroscopy. SDM is composed of two 9-aminoacridines linked via the amino groups by a spermine side chain and via the 4-positions by a N,N'-[(methylthio)ethyl]succinamide side chain [Zimmerman, S. C., Lamberson, C. R., Cory, M., & Fairley, T. A. (1989) J. Am. Chem. Soc. 111, 6805-6809]. The visible spectrum of SDM bound to poly[d(A-T)]2 or poly[d(G-C)]2 is red-shifted relative to the spectrum of SDM alone and displays considerable hypochromicity. Results from titrations of SDM with polymer indicate a binding site size of three base pairs per macrocycle. The dissociation constant for SDM bound to either poly[d(A-T)]2 or poly[d(G-C)]2 is an order of magnitude lower than that for a similar bisacridine linked only by a spermine side chain. In addition, the dependence of the dissociation constant on ionic strength is significantly reduced. NMR studies of SDM complexes with poly[d(A-T)]2 or a tetramer, d(CGCG)2, show that intercalation is the mode of binding. The magnitudes of the chemical shift differences for SDM aromatic protons in the free and bound states support intercalation with the acridine ring systems essentially parallel to the long axis of the base pairs. Cross peaks from NOESY spectra of the SDM complex with d(CGCG)2 further support this mode of binding and provide information on the structure of the complex. The results are analyzed for consistency with each of three binding models: (i) bisintercalation with the two side chains in the same groove; (ii) bisintercalation according to the neighbor-exclusion principle with the two side chains in opposite grooves; and (iii) bisintercalation with two side chains in opposite grooves but with violation of the neighbor-exclusion principle. Model i is found to be unlikely on the basis of all evidence obtained, including preliminary modeling studies. Both models ii and iii can be reconciled with the experimental evidence and from a modeling standpoint are energetically feasible.

Aminoacridines↗
PubMed1990

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.

Base Sequence↗
PubMed1990

NMR studies of an oligonucleotide with an unusual structure induced by platinum anti-cancer drugs.

The 31P NMR spectra of Pt(en)[d(T1A2T3G4G5G6T7A8C9C10C11A12T13A14)] (14-mer) and Pt(en)[d(A2T3G4G5G6T7A8C9C10C11A12T13)] (12-mer) (en = ethylenediamine) each contain two signals far downfield (ca. -2.9 and -2.6 ppm from trimethylphosphate standard), two signals slightly downfield, and at least one signal slightly upfield of the normal range (ca -4.0 to -4.4 ppm). This pattern suggested a distorted structure. The unusual 31P signals of the 12-mer were assigned by analogy to signals of the 14-mer previously assigned by 17O-labeling methods. A combination of heteronuclear multiple-quantum coherence, one-dimensional and two-dimensional nuclear Overhauser effect (1D and 2D-NOE) and homonuclear shift correlation spectroscopy (COSY) experiments assigned all aromatic 1H signals of the 12-mer except H8 of G5 or G6. One of these H8 signals is missing from the spectrum and the nucleotide is labeled Gm. The other H8 is the most downfield signal and has a strong NOE to its H1'. Since this strong NOE indicates that this nucleoside exists in a syn conformation, it is labeled Gs. A strong NOE was observed between the Gs and A8 H8 signals. Several lines of evidence suggested a hairpin-like structure with a loop region (G6T7A8C9) and a stem region involving A2T3G4G5 and C10C11A12T13. The 31P signals for the stem region are within or slightly outside the normal range. 3JH3'-P values (3-6 Hz), measured by a 2D-J experiment, of stem nucleotides were characteristic for a DNA duplex. Imino signals for base pairs A2T13, T3A12, G4C11, and probably G5C10, and the observation of internucleotide NOE connectivities for these nucleotides (e.g. between an H8 signal and the H1' signal of the 5' nucleotide) suggested a right-handed helical structure. For the loop region, a distorted sugar-phosphate backbone is indicated by far downfield positions of the G5pG6 and A8pC9 31P signals, the 3JH3'-P values for C9p (8.0 Hz) and A8p (6.8 Hz), and the absence of H3'-P coupling for G5p. In the loop region, no imino signals or internucleotide NOEs characteristic of a right-handed duplex were observed. However, A8H8, C9H6, and C10H6 each exhibited unusual internucleotide NOEs to the H4' signal of the 5' residue. NOE crosspeaks between T7 1H signals and signals attributed to sugars of the Gs and Gm suggested that the T7 moiety is located within the space encircled by the loop. The few NOE crosspeaks, pH dependence, and Cu2+ broadening of C9 1H signals indicate an isolated location accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗
PubMed1990

Sequence dependent effects in methylphosphonate deoxyribonucleotide double and triple helical complexes.

Deoxyribooligonucleotides containing 19 repeating bases of A, T or U were prepared with normal phosphodiester (dA19, dT19, dU19) or methylphosphonate (dA*19, dT*19, dU*19) linkages. Complexes of these strands have been investigated at 1:1 and 1:2 molar ratios (purine:pyrimidine) by thermal melting and gel electrophoresis. There are dramatic sequence dependent differences in stabilities of complexes containing methylphosphonate strands. Duplexes of dA*19 with dT19 or dU19 have sharp melting curves, increased Tm values, and slopes of Tm versus log (sodium ion activity) plots reduced by about one half relative to their unmodified 'parent' duplexes. Duplexes of dA19 with either dT*19 or dU*19, however, have broader melting curves, reduced Tm values at most salt concentrations and slopes of less than one tenth the values for the unmodified duplexes. Duplex stabilization due to reduced phosphate charge repulsion is offset in the pyrimidine methylphosphonate complexes by steric and other substituent effects. Triple helical complexes with dA19 + 2dT19 and dA19 + 2dU19, which can be detected by biphasic melting curves and gel electrophoresis, are stable at increased Na+ or Mg+2 concentrations. Surprisingly, however, no triple helix forms, even at very high salt concentrations, when any normal strand(s) is replaced by a methylphosphonate strand. Since triple helical complexes with methylphosphonates have been reported for shorter oligomers, inhibition with larger oligomers may vary due to their length and extent of substitution.

Base Sequence↗
PubMed1990

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs.

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.

Adenine↗