Oligonucleotide structure influences the interactions between cationic polymers and oligonucleotides

We examined the effect of oligodeoxynucleotide (ODN) structure on the interactions between cationic polymers and ODNs. Unstructured and hairpin structured ODNs were used to form complexes with the model cationic polymer, poly-L-lysine (pLL), and the characteristics of these polymer-ODN interactions were subsequently examined. We found that hairpin structured ODNs formed complexes with pLL at slightly lower pLL:ODN charge ratios as compared to unstructured ODNs and that, at high charge ratios, greater fractions of the hairpin ODNs were complexed, as measured by dye exclusion. The dissociation of pLL-ODN interactions was tested further by challenge with heparin, which induced complex disruption. Both the kinetics and heparin dose response of ODN release were determined. The absolute amount and the kinetic rate of ODN release from the complexes of pLL and unstructured ODN were greater, as compared to hairpin ODNs. Our results therefore highlight the role of ODN structure on the association-dissociation behavior of polymer-ODN complexes. These findings have implications for the selection of ODN sequences and design of polymeric carriers used for cellular delivery of ODNs.     

Oligonucleotide interactions. I. Structure of 2:1 complexes between polyuridylic acid and oligoadenylic acid

The circular dichroism of a number of 1:2 complexes between oligoadenylic acid and polyuridylic acid has been measured. The structure of these complexes, as evidenced by their optical properties, is independent of the chain length of the oligonucleotide throughout all ranges of chain lengths from adenosine to high molecular weight polyadenylic acid. A similar structure was found for the complex (Ap)₅A:2(Up)₅U.     

Effects of pD on ring-stacking interactions between dinucleoside phosphates and tryptamine

Complex formation between tryptamine and mononucleotides and dinucleoside phosphates containing adenine and/or cytosine has been studied at five pD's ranging from 1.1 to 7.4 by proton magnetic resonance spectroscopy. Chemical shifts of base ring protons and the ribose anomeric proton in the nucleotides and indole ring protons in tryptamine were monitored and their changes with pD and intermolecular interactions interpreted qualitatively. Stacked complexes were found to exist at all pD's in the range studied. Complex geometries differ depending on pD. An electrostatic interaction between the tryptamine amino group and the nucleotide phosphate group contributes to complex formation above pD 4 but is not strong enough to shift the dinucleoside phosphate equilibrium towards the unstacked conformer.     

Simple model to account for the deoxy-versus ribodimer stacking quotient data. Estimation of apparent and intrinsic equilibrium quotients for intramolecular stacking association of purine deoxy- and ribodinucleoside monophosphates

Stacking equilibrium quotients were evaluated for three purine deoxyribo-dimers, d-ApA, d-m6Apm6A and d-GpG, using procedures developed in earlier studies, and compared with those for the corresponding ribo-dimers. It was shown that the deoxyribo-dimers were apparently more strongly stacked than the corresponding ribo-dimers at 25 degrees C. To explain the observed difference in stability (as measured by the apparent stacking quotient) in the two series of dimers a new extended two-state model is advanced, that the differences in the apparent stacking quotients are due to a limited availability of the number of the sugar-ring puckering modes in the stacked ribo-dimers as compared with the case in the stacked deoxyribo-counterparts. The intrinsic stacking quotients, which are interrelated with the apparent stacking quotients by the microscopic sugar puckering equilibrium constants, were also evaluated for the purine deoxy- and ribo-dimers, and were found to be the same in any pair of these dimers.     

Calculation of the energy difference between the quaternary structures of deoxy- and oxyhemoglobin.

The energy difference between the quaternary structures of deoxy- and oxyhemoglobin is evaluated on the basis of the atomic coordinates determined by X-ray diffraction analysis. Calculation of the van der Waals interaction between subunits shows that in a hemoglobin molecule as a whole, the interaction is more attractive in the oxy form than in the deoxy form by about 8 kcal/mol, and that in each pair of two subunits except the pair alpha1alpha2, the interaction energy varies by about 15 kcal/mol. The electrostatic interactions originating in the partial charges on all constituent atoms of hemoglobin and in the polar residues on the surface of hemoglobin make only a small contribution to the energy difference between the quaternary structures of deoxy- and oxyhemoglobin. Thus, the contribution of the clusters of the polar residues in the internal cavity between like subunits and also of the freedom of rotation of the C-terminal of each subunit in oxyhemoglobin may be important energetically in the transition from deoxy to oxy quaternary structure. In this point, the present calculation supports Perutz' model, but suggests necessity of further investigations on the transitional characteristics of the quaternary structure in the intermediate steps of oxygenation. The discussion on the transitional characteristics is given in the last section.     

Conformational differences between sn-3-phospholipids and sn-2-phospholipids. A neutron and x-ray diffraction investigation

Extensive work has been reported on the conformation in membranes of sn-3-phosphatidylcholines, -ethanolamines, -glycerols and -serines (sn-3-phospholipids), where the headgroup is linked to the third carbon atom in the glycerol backbone. One important feature common to all these naturally occurring phospholipids is that the glycerol moiety is oriented almost perpendicular to the membrane surface, with the sn-1 chain continuing in this direction, whereas the sn-2 chain starts first in a direction parallel to the layer and then bends sharply at the second carbon atom. We present here neutron diffraction results on 1,3-dipalmitoyl-glycero-2-phosphocholine (1,3-DPPC) in which the headgroup is attached to the second carbon atom in the middle of the glycerol part and the two other adjacent carbon atoms are linked to the paraffin chains. Two 1,3-DPPC samples. ²H-labelled at different positions, were studied. One sample was deuterated at the first methylene segment in each fatty acyl chain, and the other at the first segment in one chain and at the second segment in the other chain. The Patterson maps as well as the neutron density profiles show that both fatty acyl chains in 1,3-DPPC have the same conformation introduced by this symmetric chemical bond pattern. It is concluded from this that the C₍₁₎C₍₃₎ vector of the glycerol backbone part is oriented parallel to the bilayer surface and the ²H nuclear magnetic resonance signals indicate that both chains have a conformation similar to that observed for the bent chain in sn-3-phospholipids. These findings indirectly confirm the idea that an intramolecular energy minimum, together with the packing geometry of the lipids in the membrane, produce the characteristic conformation around the glycerol backbone as is found for all naturally occurring phospholipids that have been studied so far.     

Proton magnetic resonance investigation of interactions between L-tryptophan and dinucleoside phosphates at low pD

Interactions between mononucleoside and dinucleoside phosphates containing adenine and/or cytosine and L -tryptophan have been studied at low pD by proton magnetic resonance (pmr) spectroscopy. The results of those studies indicate that, despite extensive protonation of ring positions, and resulting electrostatic repulsion, ring stacking does occur between both like and unlike molecules. Geometries for stacked complexes are proposed and the extent of complex formation between L -tryptophan and adenosine or cytidine in 3′ or 5′ esterified positions is discussed qualitatively.     

Conformational and dynamic differences between actin filaments polymerized from ATP- or ADP-actin monomers

Conformational and dynamic properties of actin filaments polymerized from ATP- or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys(374) residue of actin was smaller in filaments from ADP-actin than in filaments from ATP-actin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys(374) from the longitudinal filament axis. Temperature-dependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys(374) of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.     

Oligonucleotide interactions. 3. Circular dichroism studies of the conformation of deoxyoligonucleotides

The circular dichroism (CD) spectra of the four usual deoxymononucleosides, all sixteen deoxydinucleotides, and a number of trinucleotides have been measured. The dimer spectra are quite different from the sum of the spectra of their constituent monomers. This indicates the presence of base-stacked conformations analogous to those found for ribonucleoside diphosphates. The CD spectra of several deoxytrinucleotide diphosphates and single-strand f 1 DNA can be calculated fairly well by using a semi-empirical nearest-neighbor approach. There is little or no effect of terminal phosphate or of salt concentration on the optical properties of most deoxy oligomers. The possibility of simultaneous analysis of mixtures of deoxypurine or deoxypyrimidine sequence isomers has been examined. This seems to be a viable approach for the analysis of purine runs but cannot promise much success for pyrimidine runs.     

Near-heme histidine residues of deoxy- and oxymyoglobins.

Proton NMR titration curves of the histidine Cepsilon-H resonances of the deoxy and oxy forms of human, horse, and sperm whale myoglobins (Mb) were determined and compared with the results for the met and azide forms. One extra titrating resonance (H-8) was observed for each deoxy-Mb compared with the corresponding met-Mb, and a further extra resonance (H-9) was observed for the oxy-Mb form. These resonances correspond to the two additional resonances previously described for azide-Mb [Hayes, M., Hagenmaier, H., & Cohen, J. S. (1975) J. Biol. Chem. 250, 7461--7472]. This new evidence prompts us to reassign these resonances to the near-heme histidine residues.     

Influence of local interactions on protein structure. IV. Conformational energy studies of N-acetyl-N′-mehylamides of Ser-X- and X-Ser dipeptides

Conformational energy calculations using an empirical conformatinol energy program for peptides (ECEPP) werer carried out on 16 N-acetyl-N′-methylamides of Ser-X and X- Ser dipeptides, where X = Ala, Asn, Asn, Asp, Gly, Phe, Ser, Thr, and Val, and on Pro-Ser. As with the other dipeptides studied in this serites, intraresidue interactions found to dominate over interresidue interactions in determining conformational properties. The Ser-containing dipeptides (except for those with a pro or Gly residue) were found to have unusually low calculated bend probailities, in disagreement observations on proteins; this discrepancy probably arises becuse of sovent effects (not included in the computations). The Ser-X dipeptides were calculated to have a lower preference for bends than the X-Ser dipeptides.     

Cooperative interactions in the binding of ethidium ion to the self-complementary ribodinucleoside monophosphates CpG and GpC

Complexes exhibiting the characteristics of cooperative interactions are formed by ethidium ion and the self-complementary dinucleoside monophosphates CpG and GpC. Complex formation, observed with an ethidium ion selective electrode, can be described by an equilibrium binding model in which complexes are formed with dinucleoside:ethidium combining ratios of 2:1, 2:2, and 2:3. The total amount of ethidium bound in 2:2 and 2:3 complexes, as calculated from the model, is proportional to a circular dichroism band in CpG-ethidium spectra near 305 nm. Van't Hoff analysis of the model equilibrium constants reveals that the addition of ethidium ion to the 2:1 and 2:2 species is exothermic and that the corresponding entropy changes are large and negative. Cooperative interactions in the binding of ethidium ion and of other ligands to some natural and synthetic polymeric nucleic acids have now been observed in several laboratories, but the present work shows that the effect can arise even with nucleic acid fragments as small as dinucleosides. Apparently, a macromolecular nucleic acid is not essential for cooperative interactions.     

Genetic interactions between BLM and DNA ligase IV in human cells

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.     

Altered cellular interactions between endothelial cells and nonenzymatically glucosylated laminin/type IV collagen.

Laminin and type IV collagen are two major basement membrane glycoproteins. In previous studies it has been shown that nonenzymatic glucosylation induces structural alterations of these macromolecules and also reduces their ability to self-associate. In the present study, endothelial cells were tested for their ability to adhere and spread on nonenzymatically glucosylated laminin and type IV collagen. Adhesion and spreading were reduced when glucosylated macromolecules were used as substrates. Glucosylation-induced changes in adhesion and spreading may be an important initial event signaling other phenotypic modifications of cells in the microvasculature and may be a crucial factor in order to understand the pathogenesis of diabetic microangiopathy at the molecular level.     

Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2'-fluoronucleoside and arabinofuranosylcytosine]

A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.     

Competition between cell-substratum interactions and cell-cell interactions.

Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I. B., and Burdy, K.:J. Cell Physiol., 140:18-28, 1989), has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchorage-dependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions.     

Bilayers of dipalmitoyl-3-sn-phosphatidylcholine: Conformational differences between the fatty acyl chains

$\text{Dipalmitoyl}\text{-3-sn-}\text{phosphatidylcholine}$ is specifically deuterated at the C-2 position of the fatty acyl chains. Using deuterium magnetic resonance it is then possible to probe the chain conformation in the vicinity the polar head groups. Three separate quadrupole splittings are observed for bilayers of 1,2[2′-²H₂] $\text{palmitoyl}\text{-3-sn-}\text{phosphatidylcholine}$, indicating that the two chains behave differently. The synthesis of phosphatidylcholines each deuterated in only one chain allows the assignment of the three resonances. It is concluded that the beginnings of the two chains have orientations parallel and perpendicular to the bilayer normal. The data further suggest the possibility of two long-lived conformations of the glycerol constituent.