An AFM investigation of oligonucleotides anchored on unoxidized crystalline silicon surfaces.

Carboxylic terminated monolayers have been covalently attached on phosphorous doped crystalline (100) silicon surfaces using a cathodic electro grafting technique. The functionalization concentration and efficiency have been evaluated with different techniques. In particular, topographic images, performed with an atomic force microscope, were used to optimize the protocol in order to obtain a surface whose characteristics of uniformity and reproducibility are ideal for a bio-electronic device. Phase lag images of the functionalized surfaces were also performed, and show non-topographic structures that have been interpreted as areas of different molecule self-orientation. Poly-thymine oligonucleotides have been anchored on such a surface to form a nano-biosensing device capable to react selectively with a specific target molecule, a poly-adenine oligonucleotide. AFM images of high density (approximately 3x10(12) mol/cm2) single strand and double strand covered samples show toroidal shaped structures formed by the self-assembly of the oligonucleotides on the silicon surface.

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein.

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

Influence of an 8-oxoadenine lesion on the structural and dynamic features of a 30-mer DNA fragment with and without a mismatch.

PURPOSE: To elucidate the influence of the oxidative lesion 7,8-dihydro-8-oxoadenine (8-oxoA) on the structural and dynamic features of a 30-mer DNA fragment, and to understand if differences occur when C is positioned opposite 8-oxoA instead of T. MATERIALS AND METHODS: Two 30-mer DNA oligomers with or without the 8-oxoA and two complementary oligomers with C or T base opposite the lesion site were synthesized and annealed. Duplexes named AT, A*T, AC and A*C were characterized by means of circular dichroism and UV denaturation measurements. gamma-Ray footprinting experiments were performed to give insight into their fine three-dimensional structure. Elastic torsional constants were derived by following the decay of the fluorescence polarization anisotropy (FPA) of the ethidium-DNA complexes measured by multifrequency-phase fluorometry. RESULTS: The introduction of one oxidative lesion in a 30-mer DNA oligomer with and without a mismatch did not cause relevant changes in their overall conformation and slightly modified their elastic properties. Small energetic differences were revealed by thermodynamic analysis in the sample bearing both the oxidative lesion and the mismatch. Minor variations in the cleavage pattern due to the hydroxyl radicals in the A*T sample were observed and present along the entire DNA fragment length. In the A*C sample, by contrast, there was a major modification in the cleavage pattern extending for about 11 bases around the lesion, especially towards the 5'-end. CONCLUSIONS: Differences in the fine structure and in the elastic properties between the A*T and A*C samples were observed, while their overall conformation was unchanged. The results are consistent with the hypothesis that the observed local changes of the double helix structure in A*C are due to the pairing of the oxidized adenine in a syn conformation with the cytosine.

Analysis of efficiency and fidelity of HIV-1 (+)-strand DNA synthesis reveals a novel rate-limiting step during retroviral reverse transcription.

We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (+)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (+)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNA-primed (-)-strand DNA synthesis.

Oligonucleotide labeling: synthesis of a new spin-labeled 2'-deoxyguanosine analogue.

In order to achieve an EPR sensitive probe for DNA, 3-carboxy-Proxyl free radical was linked to O-6 of dG through a five-atoms-tether. The modified base was incorporated into a 30-mer ODN, then annealed to its complementary DNA strand. Hydrodynamic parameters show only a slight destabilization with respect to the equivalent unlabeled hybrid. EPR could monitor the hybrid formation showing a progressive enlargement of the upfield signal in passing from the labeled ss- to the ds-30-mer.

DNA, RNA and hybrid RNA-DNA oligomers of identical sequence: structural and dynamic differences.

A 27-mer sequence was synthesised as DNA duplex (DD), RNA duplex (RR), and RNA-DNA (RD) hybrid in order to characterise their structural and dynamic features. The hydrodynamic radius (Rh) and the rise (b) values of the three samples were consistent with the conformations predicted by CD analysis. The value of the torsional constant (alpha) of the samples containing RNA was approximately twice that of the DD sample and followed the order: DD < RD < RR. The same order was observed in the thermodynamic stability and in the reduction of the electrophoretic mobility. gamma-Ray footprinting analysis was carried out to resolve the individual strand conformation in the hybrid. The RNA strand preserved its conformation, while the DNA strand showed local deformations mainly at TA and TG steps.

gamma-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2'-deoxy-7-hydro-8- oxoguanosine lesion.

The influence of the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5'-GATCCTCTAGAGTC[G(*) or G]ACCTGCAGGCATGCA-3']:[3'-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5'], in which G(*) is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G(*)C and G(*)A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar gamma-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G(*) which are extended for almost one helical turn. Some differences are observed between G(*)C and G(*)A duplexes. In particular, in the G(*)C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G(*)A sample a shift of the minimum is observed. The hydrodynamic radius R(h) derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 +/- 0.2 A between 5 and 40 degrees C, in all the samples. The torsional constant alpha of each oligomer decreases when the temperature is raised and the alpha values of the damaged samples are higher than those of the normal ones.

NMR structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) comprising the tRNA-DNA junction formed during initiation of HIV-1 reverse transcription.

A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA-DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5 A in the RNA/RNA stem to about 4.5 A in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of approximately 11 A and approximately 8.5 A, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in 'induced-fit'-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex.

"Back door" opening implied by the crystal structure of a carbamoylated acetylcholinesterase.

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up.

The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding

Temporal coordination between initiation of HIV (+)-strand DNA synthesis and primer removal.

In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using an in vitro system that closely mimics the initiation of (+)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3'- and 5'-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5' direction of the RNA strand. DNA synthesis resumes again in the 3' direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position +12, by treating these with Fe2+ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (+)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex.

Fractions of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped viruses: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus.

A series of chemically oversulphated galactosaminoglycans (SO3H:COOH ratio > or = 2) were tested in vitro as antiviral agents against human immunodeficiency virus type 1 (HIV-1), the aetiological agent of AIDS, and against herpes simplex virus type 1 and human cytomegalovirus, two agents responsible for opportunistic infections in HIV-infected people. The oversulphated derivatives displayed an increase in activity ranging from one to four orders of magnitude against the three viruses, as compared to the natural parent compounds (SO3H:COOH, ratio approx. 1). The antiviral activity of these polyanions appears to be favoured by a high degree of sulphation and a high molecular mass. An oversulphated dermatan, with a SO3H:COOH ratio of 2.86 and molecular mass of 23.2 kDa, was the most potent anti-HIV-1 compound (EC50 0.04 microgram/ml). A second oversulphated dermatan, with a SO3H:COOH ratio of 2.40 and molecular mass of 25 kDa, displayed the highest activity against HSV-1 (EC50 0.01 microgram/ml). An oversulphated chondroitin, with a SO3H:COOH ratio of 2.80 and molecular mass of 17.3 kDa, was the strongest anti-HCMV agent (EC50 0.4 microgram/ml). In view of the absence of the side-effects typical of heparin-like compounds, a combination of these derivatives could have therapeutic potential.

Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode.

Single-turnover and equilibrium measurements were carried out to determine the basis of the apparently slow, nonprocessive polymerization reaction catalyzed by HIV-1 reverse transcriptase (RT) during transcription initiation, when both the primer and template are composed of RNA. Comparison of the binding and kinetic parameters of a 20-mer, all-RNA primer/35-mer template substrate to one identical in sequence but composed of a 20-mer, all-DNA primer/35-mer RNA template reveals striking differences. Equilibrium titrations yielded a dissociation constant (Kd) >200 nM for the RNA/RNA-RT complex which is at least 200-fold higher than that of the DNA/RNA-substrate (Kd approximately 1 nM). The affinity of the RT-RNA/RNA complex for dTTP was found to be at least 500 times lower (Kd approximately 3.4 mM) than that of the RT-DNA/RNA complex (Kd approximately 6.6 microM). The single-turnover dNTP incorporation time course using the RNA-primer substrate, the DNA-primer substrate, or a series of RNA-primer substrates preextended with one to eight deoxynucleotides showed that dNTP incorporation occurs with a biphasic exponential burst of +1 extension product, followed by a linear phase. At least three different RT-bound forms of the p/ts exist: a fast, kinetically competent form (single-turnover rate approximately 10-50 s-1); a slow form (rate approximately 0.3-1 s-1); and a form that is dead-end (no turnover). The studies further revealed that a switch to a fast, kinetically competent p/t occurs after six dNTPs are incorporated into the RNA primer, with the switch being defined as the transition from a minority to a majority of the p/t bound in the optimal manner.

3-(Carboxyalkylthio) rifamycin S and SV derivatives inhibit human neutrophil functions.

In order to further investigate the potential of rifamycins as antiinflammatory drugs, twenty-five semisynthetic rifamycins were tested at concentrations ranging from 10(-9) to 10(-5) M on in vitro human neutrophil functions such as locomotion, superoxide anion production, and degranulation, under different stimulatory conditions. They were also tested as antiproliferative agents on peripheral blood lymphocytes. The present semisynthetic derivatives are in general characterized by their carrying a hydrophilic substituent; they are rifamycin S or rifamycin SV derivatives carrying at C(3) either a carboxyalkyl side-chain or a glycosyl side-chain. Derivatives of the former group displayed inhibitory activities covering the whole range of activities tested, suggesting that the sum of these different effects could support their antiinflammatory activity in vivo. These derivatives, carrying a free carboxyl, are more water soluble than rifamycin SV at physiological pH, and may serve as antiinflammatory drugs for local administration, alternative to rifamycin SV, possibly giving higher efficacy and reduced side effects of pain and tissue swelling.

Crystal structure of an eight-base pair duplex containing the 3'-DNA-RNA-5' junction formed during initiation of minus-strand synthesis of HIV replication.

During initiation of minus-strand synthesis by HIV-1 reverse transcriptase, a 3'-DNA-RNA-5' junction is formed involving the 3'-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the RNA template strand specifically, opposite the newly synthesized DNA strand. We have determined the crystal structure at 1.9 A resolution of an eight-base pair hybrid duplex representing the junction to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. The junction octamer is in a global A-type conformation throughout. A base pair step with distinct stacking geometry and variable backbone conformation is located next to the main endonucleolytic cleavage site. This base pair step may serve as a recognition site for HIV-RT RNase H.

Synthesis and activity studies of N-[omega-N'-(adamant-1'-yl)aminoalkyl]- 2-(4'-dimethylaminophenyl)acetamides: in the search of selective inhibitors for the different molecular forms of acetylcholinesterase.

A series of N-[omega-N'-(adamant-1'-yl)aminoalkyl]-2-(4'- dimethylaminophenyl)acetamides were synthesized and tested as acetylcholinesterase inhibitors. A significant selectivity toward acetylcholinesterases from various natural sources, mainly differing in their quaternary structure and solubility, was pointed out. The interest of this kind of molecules as potential therapeutic agents for Alzheimer's disease is discussed.

Capillary electrophoretic analysis of synthetic short-chain oligoribonucleotides.

Thirty synthetic oligoribonucleotides, 3 to 18 nucleotides (nt) long, were analyzed by capillary electrophoresis, under nondenaturing conditions, using a commercial kit. The migration time t(m) was dependent on nt length and composition, capillary length, operating temperature, and type of sieving polymer. Under fixed experimental conditions, the t(m) proved predictable by the equation: t(m) = [0.22(n-1) + 6.14A/n + 6.86G/n + 3.61 (C+U)/n] min, for n>3, where A/n, G/n, C/n, U/n is the frequency of each type of nt within the oligonucleotide (ONT). The equation accounts for the influence of charge-to-mass ratio on t(m), but not for structural effects, if present. This approximation is acceptable for short ONTs. The possibility of detecting n+1, n-1, n-2 impurities, having predicted the t(m), is of crucial importance in assessing the purity of synthetic ONTs dedicated to structural studies. This appears to be feasible. High resolution was shown among homologous series of ONTs of increasing length, and in some cases, even within groups of ONTs of the same length but different composition. The addition of 7 M urea to the buffer, as denaturing agent, accelerates the t(m) and significantly lowers the resolution for the shortest ONTs. It was also possible to monitor the state of association of mixtures of RNA and DNA sequence-complementary strands.

Rifamycins inhibit human neutrophil functions: new derivatives with potential antiinflammatory activity.

In our study we investigated the effect of rifamycin SV, rifamycin B, rifampicin and five semisynthetic derivatives on human neutrophil functions such as locomotion, superoxide production and degranulation stimulated by specific agonists. All compounds were tested at concentrations ranging from 10(-9) to 10(-5) M. Among the newly synthesized compounds the most active we found to be the derivatives carrying an acidic substituent at C3: these significantly lowered the superoxide generation induced by PMA throughout the entire concentration range, whereas rifamycin SV, rifamycin B and rifampicin were effective only at the highest concentrations. Moreover, chemotactic movement was significantly attenuated by derivative R4, rifamycin B and rifamycin SV at high doses; granule enzyme release was unaffected by all compounds.

Long chain analogs of physostigmine as potential drugs for Alzheimer's disease: new insights into the mechanism of action in the inhibition of acetylcholinesterase.

Heptylphysostigmine is in advanced clinical trial as a drug for Alzheimer's disease. 8-Morpholinooctylphysostigmine and 8-(cis-2,6-dimethylmorpholino)octylphysostigmine are currently undergoing pre-clinical evaluation. The mechanism of action of these compounds in the inhibition of acetylcholinesterase has been investigated. All the examined compounds display non competitive-like kinetics of inhibition. There are no reversible components in the observed inhibition: the whole inhibitory effect is due to the time-dependent pseudo-irreversible carbamylation of the active site. Yet the observed time course of the inhibition does not match a simple second order kinetics. An influence of the quaternary structure of the enzyme on the more complex kinetics of carbamylation is hypothesized. Reactivation experiments on the inhibited enzyme show long lasting inhibitory effects for these compounds. The higher duration of the anticholinesterase effect of the morpholino derivatives compared to heptylphysostigmine should provide the basis for their higher therapeutic potential.