Islet surface modification with urokinase through DNA hybridization

Transplantation of islets of Langerhans (islets) has been proposed as a safe, effective approach to treating patients with insulin-dependent diabetes mellitus (type I diabetes). It has been reported, however, that many islets are lost in the early phase after intraportal transplantation by instant blood coagulation-mediated inflammatory reactions. In this study, DNA hybridization was applied to conjugate the fibrinolytic enzyme urokinase on the islet surface. We synthesized amphiphilic polymers, PEG-lipids carrying oligo(dT)(20) (oligo(dT)(20)-PEG-lipid; PEG MW = 5000) and urokinase (UK) carrying oligo(dA)(20). The oligo(dT)(20)-PEG-lipid was spontaneously incorporated into the cell membrane through interactions between the hydrophobic parts of the PEG-lipids and the lipid bilayer, and UK was conjugated on the cell surface through DNA hybridization between oligo(dT)(20) on the cell and complementary oligo(dA)(20) on the UK. The activity of UK was maintained on the islet surface. The surface modification with UK did not influence islet morphology or islet ability to secrete insulin in response to changes in glucose concentration. No practical volume increase was observed with our method, indicating that islet graft loss could be suppressed at the early stage of intraportal islet transplantation.     

Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ was studied by UV–vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV–vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.     

CD studies on the conformation of some deoxyoligonucleotides containing adenine and thymine residues

CD studies of the deoxyoligomer series d(pT)_n and d(pA)_n show increasing CD maxima for oligo (dT)'s with chain length variation from two to seven, while oligo (dA)'s exhibit a decreasing CD maximum. Concentrated solutions of NaClO₄ cause a decrease in the CD of longer oligo (dT)'s towards the CD of d(pT)₂ which is different from oligo dA's. Probably base-sugar interactions are important in the observed conformational effects. The chemically synthesized oligomers dpApApTpT and dpTpApTpA show deviations in their CD spectra which reflect a dominating conformational effect of d(pA)₂ in the former but not in the alternating isomer.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.     

Studies on the processivity of highly purified calf thymus 8S and 7.3S DNA polymerase alpha

Template-challenge experiments indicate no gross difference in processivity of the calf thymus DNA polymerase α A and C enzymes. Both enzymes appear to be distributive. Results showing the apparent processive nature of both enzymes on poly (dC). oligo (dG)₁₀ when challenged with poly (dA). oligo (dT)₁₀ are explicable by the failure of both enzymes to bind to the challenging template rather than by the presence of an initiation factor which preferentially binds to certain templates.     

Mechanistic study of E. coli DNA topoisomerase I: cleavage of oligonucleotides.

E. coli DNA topoisomerase I catalyzes DNA topoisomerization by transiently breaking and rejoining single DNA strands (1). When an enzyme-DNA incubation mixture is treated with alkaline or detergent, DNA strand cleavage occurs, and the enzyme becomes covalently linked to the 5'-phosphoryl end of the cleaved DNA (2). Using oligonucleotides of defined length and sequence composition, this cleavage reaction is utilized to study the mechanism of E. coli DNA topoisomerase I. dA7 is the shortest oligonucleotide tested that can be cleaved by the enzyme. dT8 is the shortest oligo(dT) that can be cleaved. The site of cleavage in both cases is four nucleotides from the 3' end of the oligonucleotide. No cleavage can be observed for oligo(dC) and oligo(dG) of length up to eleven bases long. dC15 and dC16 are cleaved at one tenth or less the efficiency of oligo(dA) and oligo(dT) of comparable length.     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.     

Berenil Complexation with a Nucleic Acid Triple Helix

Abstract

The interaction of berenii molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)₁₂?x-(dT)₁₂?x-(dT)₁₂, where x is a hexaethylene glycol chain bridged between the 3′ phosphate of one strand and the 5′ phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that berenil can bind to the triple helical structure. Spectral analysis shows that in the same ionic strength the drug bound to a double-stranded structure exhibits a conformation and an environment close to those observed in triple-stranded structure. The influence of the ionic strength on the interaction between the berenil molecule and the 36-mer is clearly demonstrated. We showed that when no NaCl salt is added in the buffer the triplex form of (dA)₁₂?x-(dT)₁2-x-(dT)₁₂ is stabilized by berenil whereas it is destabilized slightly by the dye when NaCl concentration is 1 M.     

The Unusual X-Form DNA in Oligodeoxynucleotides: Dependence of Stability on the Base Sequence and Length

Abstract

X-form is an unusual double helix of DNA adopted by poly(dA-dT) or (dT-dA)₄ at high concentrations of CsF. On the other hand, poly(dA).poly(dT), (dA-dT)₄ and most other DNAs do not adopt this conformer. Here we demonstrate that the X-form is strongly destabilized by GC pairs or even minute perturbations of the alternating pyrimidine- purine sequence. For example, the 30-mer d(TATAAT)₅, containing five tandem repeats of the Pribnow box, fails to isomerize into the X-form. After (dT-dA)₄, the 16-mer (dT- dA)_g is shown to be the second most predisposed oligodeoxynucleotide in the (dT-dA)., series to isomerize into the X-form while the duplex lengths corresponding to n=3,5,6,7,9,12 and 20 make the X-form unstable even in the strictly alternating (dT-dA)., sequence. Consequently, the (dT-dA)., duplex length is also a crucial factor of the X-form stability on the oligodeoxynucleotide level. We discuss a possibility that the X-form is a solution counterpart of the D-form adopted in dehydrated poly(dA-dT) fibers because properties of these two conformers are remarkably similar in many respects.     

Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.     

A UV Resonant Raman Spectroscopic Study of the Interaction of Metallic Derivatives of Tetrakis(4-N-methylpyridyl)porphine with Polynucleotides

Abstract

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)₂, poly(dA)poly(dT), poly(dG.dC)₂ and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC)₂. Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.     

Liposomes as carriers of the lipid soluble antioxidant resveratrol: Evaluation of amelioration of oxidative stress by additional antioxidant vitamin

Aim

Resveratrol (RES) is a well-known antioxidant, yet in combination with other antioxidant vitamins, it was found to be more effective than any of these antioxidants alone. Present work aims to compare the antioxidant actions of resveratrol with and without vitamin C following delivery as liposomes tested using chemical and cellular antioxidative test systems.

Main methods

Liposomes were prepared by the thin film hydration method and characterised for percent drug entrapment (PDE), Z-average mean size (nm), polydispersity index (PDI) and zeta potential. Antioxidative capacity was determined by studying the inhibition of AAPH induced luminol enhanced chemiluminescence and inhibition of ROS production in isolated blood leukocytes. Intracellular oxygen-derived radicals were measured using flow cytometry with buffy coats (BC) and human umbilical vein endothelial cells using H₂DCF-DA dye.

Key findings

Particle size varied from 134.2 ± 0.265 nm to 103.3 ± 1.687 nm; PDI ≤ 0.3; zeta potential values were greater than − 30 mV and PDE ≥ 80%. Radical scavenging effect was enhanced with liposomal systems; oxidative burst reaction in BC was inhibited by liposomal formulations, with the effect slightly enhanced in presence of vitamin C. Reduction in reactive oxygen species (ROS) production during spontaneous oxidative burst of BC and incubation of HUVECs with H₂O₂ further intensified the antioxidative effects of pure RES and liposomal formulations.

Significance

The present work clearly shows that the antioxidative effects of resveratrol loaded into liposomes are more pronounced when compared to pure resveratrol. Liposomal resveratrol is even active within the intracellular compartments as RES could effectively quench the intracellular accumulation of ROS.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.