Improved thrombin binding aptamer analogues containing inversion of polarity sites: structural effects of extra-residues at the ends.

In this paper, we report the investigations, based on NMR, molecular modelling, CD measurements and electrophoresis, of thrombin binding aptamer (TBA) analogues containing an extra-residue at the 3'-end or at both the ends of the original TBA sequence, linked through 3'-3' or 5'-5' phosphodiester bonds. The data indicate that most of the modified aptamers investigated adopt chair-like G-quadruplex structures very similar to that of the TBA and that stacking interactions occur between the 3'-3' or 5'-5' extra residues and the deoxyguanosines of the upper G-tetrad. A comparison of the thermodynamic data of TBA-A and TBA-T containing a 3'-3' extra residue and their canonical versions clearly indicates that the 3'-3' phosphodiester bond is fundamental in endowing the modified aptamers with remarkably higher thermal stabilities than the original TBA.

A novel pyrimidine tetrad contributing to stabilize tetramolecular G-quadruplex structures.

G-quadruplex structures formed by oligodeoxyribonucleotides TGGU(NH2)GGT (AM, U(NH2) = 5-amino-2'-deoxyuridine), TGGU(Br)GGT (BR, U(Br) = 5-bromo-2'-deoxyuridine) and TGGTGGT (TH) have been investigated through circular dichroism, nuclear magnetic resonance, gel electrophoresis and molecular modeling techniques. Collected data indicate that all 7-mer oligonucleotides form tetramolecular parallel G-quadruplex structures with all residues adopting anti glycosidic bonds. In the case of AM, data suggest the occurrence of a novel U(NH2)-tetrad characterized by eight hydrogen bonds that stabilizes the G-quadruplex structure more efficiently than U(Br)- and T-tetrads.

Detection of potato brown rot and ring rot by electronic nose: from laboratory to real scale.

A commercial electronic nose (e-nose) equipped with a metal oxide sensor array was trained to recognize volatile compounds emitted by potatoes experimentally infected with Ralstonia solanacearum or Clavibacter michiganensis subsp. sepedonicus, which are bacterial agents of potato brown and ring rot, respectively. Two sampling procedures for volatile compounds were tested on pooled tubers sealed in 0.5-1 L jars at room temperature (laboratory conditions): an enrichment unit containing different adsorbent materials (namely, Tenax(®) TA, Carbotrap, Tenax(®) GR, and Carboxen 569) directly coupled with the e-nose (active sampling) and a Radiello(™) cartridge (passive sampling) containing a generic Carbograph fiber. Tenax(®) TA resulted the most suitable adsorbent material for active sampling. Linear discriminant analysis (LDA) correctly classified 57.4 and 81.3% total samples as healthy or diseased, when using active and passive sampling, respectively. These results suggested the use of passive sampling to discriminate healthy from diseased tubers under intermediate and real scale conditions. 80 and 90% total samples were correctly classified by LDA under intermediate (100 tubers stored at 4°C in net bag passively sampled) and real scale conditions (tubers stored at 4°C in 1.25 t bags passively sampled). Principal component analysis (PCA) of sensorial analysis data under laboratory conditions highlighted a strict relationship between the disease severity and the responses of the e-nose sensors, whose sensitivity threshold was linked to the presence of at least one tuber per sample showing medium disease symptoms. At intermediate and real scale conditions, data distribution agreed with disease incidence (percentage of diseased tubers), owing to the low storage temperature and volatile compounds unconfinement conditions adopted.

In vivo assessment of CdSe-ZnS quantum dots: coating dependent bioaccumulation and genotoxicity.

Semiconductor nanocrystals, or Quantum Dots (QDs), have gained considerable attention due to their unique size-dependent optical and electronic properties that make them attractive for a wide range of applications, including biology and nanomedicine. Their widespread use, however, poses urgent questions about their potential toxicity, especially because of their heavy metal composition that could cause harmful effects to human health and environment. In this work, we evaluated in vivo the long-term toxicity of CdSe-ZnS QDs with different surface coatings, probing oral administration in the model system Drosophila melanogaster. In particular, we found that all the differently coated QDs significantly affect the lifespan of treated Drosophila populations and induce a marked increase in reactive oxygen species (ROS) levels. Furthermore, we observed that these QDs induce severe genotoxic effects and increased rate of apoptosis in Drosophila haemocytes. These toxic effects were found to be mainly related to the in vivo degradation of QDs with consequent release of Cd(2+) ions, while the coating of QDs can modulate their bioaccumulation in the organism, partly decreasing their overall toxicity.

Physical assessment of toxicology at nanoscale: nano dose-metrics and toxicity factor.

In this work, we propose a systematic and reproducible evaluation of nanoparticles (NPs) toxicology in living systems, based on a physical assessment and quantification of the toxic effects of NPs by the experimental determination of the key parameter affecting the toxicity outcome (i.e., the number of NPs) and of the NPs "toxicity factor". Such a strategy was applied to a well determined scenario, i.e., the ingestion of citrate-capped gold NPs (AuNPs) of different sizes by the model system Drosophila melanogaster. Using these AuNPs as a reference toxicity standard, we were able to define different regions in the multiparametric space of toxicity, enabling the classification of the toxic levels of other nanomaterials, such as quantum dots and pegylated AuNPs. This approach may pave the way to a systematic classification of nanomaterials, leading to important developments in risk assessment and regulatory approval, as well as in a wide range of nanomedicine applications.

Studies on the influence of inversion of polarity sites on the dG residues glycosidic conformation in quadruplex structures.

Insights into the influence of inversion of polarity sites on the dG residues glycosidic conformation in quadruplexes is presented. The NMR studies concern modified oligodeoxynucleotides based on the quadruplex forming sequence TGGGT.

A mini-library of TBA analogues containing 3'-3' and 5'-5' inversion of polarity sites.

Several researches have been devoted to structure-activity relationship and to post-SELEX modifications of the thrombin binding aptamer (TBA), one of the first aptamers discovered by the SELEX methodology. However, no studies on TBA dealing with the effects of introduction of inversion of polarity sites have been reported yet. In this frame, we have undertaken the synthesis and the study of a mini-library composed of several TBA analogues containing a 3'-3' or a 5'-5' inversion of polarity site at different positions into the sequence. Particularly, in this article, we present preliminary results about their structural and biological properties.

Effect of the introduction of an A-residue into a quadruplex forming oligonucleotide containing a 5'-5' polarity of inversion site.

Preliminary NMR studies on structure formed by sequence 3'-TGA-5'-5'-GGT-3' are described. We proposed the formation of a tetramolecular quadruplex in which strands are equivalent to each other and three G-tetrads are present. The possibility of the occurrence of an A-tetrad also is discussed.

A topological classification of G-quadruplex structures.

A topological classification of most quadruplex structures is proposed, based on two main characteristics: 1) the relative orientation of the strands and 2) the nature of the loops connecting the strands.

Synthesis of 5-methylamino-2'-deoxyuridine derivatives.

Reductive amination of 3',5'-O-(tetraisopropyldisilyloxane-1,3-diyl)-2'-deoxy-5-formyluridine with several aliphatic and aromatic amines, in various solvents, is described. In the case of aliphatic amines, the expected C-5 substituted methylamino pyrimidine nucleosides are formed along with by-products deriving from opening of the pyrimidine ring. Relative amounts of the by-products depend upon the polarity of the solvent employed.

Solid-phase synthesis of oligonucleotides containing a bipyridine ligand at the 3'-3' inversion of polarity site.

The preparation of a solid support useful for the synthesis of oligonucleotides with a 3'-3' inversion of polarity, via a linker containing a chelating molecule, namely 2,2'-bipyridine, is described.

2'-Deoxy-8-(propyn-1-yl)adenosine-containing oligonucleotides: effects on stability of duplex and quadruplex structures.

2'-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its influence on thermal stability of duplex and quadruplex structures investigated by UV, CD and 1H NMR. The obtained results seem to indicate that the presence of the modified base negatively affects the stability of double stranded DNA whereas remarkably increases the stability of parallel quadruplex structures.

Synthesis and characterisation of poly(D,L-lactic acid)-idoxuridine conjugate.

A new polymeric prodrug was prepared coupling 5-iodo-2'-deoxyuridine (IDU) to poly(d,l-lactic acid) (PLA) via a succinic acid spacer. The PLA-IDU conjugate was characterised by thermal analysis, IR and 1H and 13C NMR spectroscopy. The IDU content (0.024 mequiv.g-1 of PLA) was consistent with the carboxylic acid endgroup present in the polymer sample (0.025 mequiv.g-1 of polymer). The PLA-IDU conjugate was susceptible to degradation in biological environments containing esterase, whereas IDU was not detected by chemical hydrolysis in pH 7.4 phosphate buffer. The conjugate should be used to prepare injectable microspheres and nanospheres containing IDU chemically coupled to the polymer carrier.

Affinity, stability and polarity of binding of the TATA binding protein governed by flexure at the TATA Box.

The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable ("committed") transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.

Design and NMR study of an immobile DNA four-way junction containing 38 nucleotides.

The DNA Holliday junction is a central intermediate in genetic recombination. We have designed and synthesized a DNA oligomer, J1a, as a model compound for the Holliday junction suitable to be studied by NMR spectroscopy and future molecular modelling. The design was based on a 46-base oligomer, J4, previously studied by Pikkemaat, J. A., van den Elst, H., van Boom, J. H. & Altona, C. [Biochemistry 33, 14896-14907 (1994)], including the propensity to undergo a self-folding process to give a four-way junction in which three of the four arms are capped with a hairpin loop. J1a, however, is considerably shortened by eight bases and thus contains only 38 residues which significantly facilitates the proton resonance assignments. The base sequence at the branch point is identical to that in J4. 1H-NMR data clearly point to the presence of three hairpin loops in J1a and show that the double-helical arms adopt the B-DNA form. Quasicontinuous pairwise stacking between helical arms to give a single preferred stacked X-conformation is evident. The extent of folding into this stacked conformation is strongly dependent upon the magnesium concentration. Full Watson-Crick base pairing at the branch point is completely preserved. The A/D-stacking preference of the small junction is the same as that exhibited by J4.

Twin hydroxymethyluracil-A base pair steps define the binding site for the DNA-binding protein TF1.

The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1-encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (Kd was approximately 3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based largely, if not entirely, on sequence-dependent DNA flexibility.

1H NMR studies of the 5-(hydroxymethyl)-2'-deoxyuridine containing TF1 binding site.

The pyrimidine base 5-(hydroxymethyl)-2'-deoxyuridine (HmU) is a common nucleotide in SPO1 phage DNA. Numerous transcriptional proteins bind HmU-containing DNA preferentially implicating a regulatory function of HmU. We have investigated the conformation and dynamics of d-(5'-CHmUCHmUACACGHmUGHmUAGAG-OH-3')2 (HmU-DNA). This oligonucleotide mimics the consensus sequence of Transcription Factor 1 (TF1). The HmU-DNA was compared to the thymine-containing oligonucleotide. NOESY and DQF COSY spectroscopy provided resonance assignments of nonexchangeable and exchangeable protons, intranucleotide, internucleotide and intrastrand proton-proton distances, and dihedral angle constraints. Methylene protons of the hydroxymethyl group are nonequivalent protons and the hydroxymethyl group is not freely rotating. The hydroxymethyl group adopts a specific orientation with the OH group oriented on the 3' side of the plane of the base. Analysis of imino proton resonances and NOEs indicates additional end base pair fraying and a temperature-induced transition to a conformation in which the internal HmU-A base pairs are disrupted or have reduced lifetimes. Orientation of the hydroxymethyl group indicates the presence of internucleotide intrastrand hydrogen bonding between the HmU12C5 hydroxyl group and A13. All sugars in both DNAs show a C2'endo conformation (typical of B-DNA).

Localized DNA flexibility contributes to target site selection by DNA-bending proteins.

Certain DNA-binding proteins function as architectural elements by bending DNA. We have studied the binding of three such proteins, the prokaryotic HU and integration host factor (IHF) and the eukaryotic HMG1, to DNA in which flexibility is enhanced by tandem mismatches and by substituting 5-hydroxymethyluracil (hmU) for thymine (T). IHF and HU have higher affinity for DNA with two 4-nt loops than for perfect duplex DNA with a sequence that corresponds to a binding site for the phage-encoded homolog, TF1. HU has a high affinity for DNA with 4-nt loops separated by 9 bp (Kd = 3.5 nM), with suboptimal binding for other loop separations. IHF-binding is optimal when 4-nt loops are 8 to 9 bp apart; optimal complex formation with DNA representing the specific IHF-binding site H' requires that loops do not disrupt the consensus sequence and that one 4-nt loop borders the dyad axis-proximal block of consensus sequence (Kd = 0.3 nM, approximately tenfold lower than for H' perfect duplex DNA). HMG1 also binds preferentially to DNA with loops. All three proteins bind more tightly to DNA in which thymine is replaced with hmU. IHF has a tenfold higher affinity for hmU-DNA without a consensus IHF site (Kd = 7.6 nM) than for the corresponding T-DNA but does exhibit site-selectivity in hmU-DNA; Kd = 0.6 nM for the hmU-containing version of H'. Tighter binding to hmU-DNA is consistent with greater flexibility, and the distinct influence of loop position on complex formation suggests that sequence-dependent variations in flexibility of duplex DNA play a significant role in target-site selection by these DNA-bending proteins.

On the connection between inherent DNA flexure and preferred binding of hydroxymethyluracil-containing DNA by the type II DNA-binding protein TF1.

TF1 is a member of the family of type II DNA-binding proteins, which also includes the bacterial HU proteins and the Escherichia coli integration host factor (IHF). Distinctive to TF1, which is encoded by the Bacillus subtilis bacteriophage SPO1, is its preferential binding to DNA in which thymine is replaced by 5-hydroxymethyluracil (hmU), as it is in the phage genome. TF1 binds to preferred sites within the phage genome and generates pronounced DNA bending. The extent to which DNA flexibility contributes to the sequence-specific binding of TF1, and the connection between hmU preference and DNA flexibility has been examined. Model flexible sites, consisting of consecutive mismatches, increase the affinity of thymine-containing DNA for TF1. In particular, tandem mismatches separated by nine base-pairs generate an increase, by orders of magnitude, in the affinity of TF1 for T-containing DNA with the sequence of a preferred TF1 binding site, and fully match the affinity of TF1 for this cognate site in hmU-containing DNA (Kd approximately 3 nM). Other placements of loops generate suboptimal binding. This is consistent with a significant contribution of site-specific DNA flexibility to complex formation. Analysis of complexes with hmU-DNA of decreasing length shows that a major part of the binding affinity is generated within a central 19 bp segment (delta G0 = 41.7 kJ mol-1) with more-distal DNA contributing modestly to the affinity (delta delta G = -0.42 kJ mol-1 bp-1 on increasing duplex length to 37 bp). However, a previously characterised thermostable and more tightly binding mutant TF1, TF1(E15G/T32I), derives most of its extra affinity from interaction with flanking DNA. We propose that inherent but sequence-dependent deformability of hmU-containing DNA underlies the preferential binding of TF1 and that TF1-induced DNA bendings is a result of distortions at two distinct sites separated by 9 bp of duplex DNA.

Slow conformational exchange in DNA minihairpin loops: a conformational study of the circular dumbbell d.

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson-Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decamer 5'-d-3' (I) is studied in solution by means of nmr spectroscopy. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson-Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5'-GTTC-3' loop with only one remaining solvent-accessible hydrogen bond between NH alpha of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5'-CTTG-3' loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5'-3' loop-stem junction in the 5'-GTTC-3' loop. The two conformers, L2L2 and L2L4, occur in slow equilibrium (2-20 s-1). Moderate line broadening of specific 1H, 13C, and 31P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T1 relaxation measurements, chemical-shift considerations, and complete band-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational states in the anti-syn equilibrium.