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.

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.

Outstanding effects on antithrombin activity of modified TBA diastereomers containing an optically pure acyclic nucleotide analogue.

Herein, we report optically pure modified acyclic nucleosides as ideal probes for aptamer modification. These new monomers offer unique advantages in exploring the role played in thrombin inhibition by a single residue modification at key positions of the TBA structure.

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.

NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA.

Incorporation of 5-(hydroxymethyl)-2'-deoxyuridine into DNA in place of thymine by SPO1, a Bacillus subtilis bacteriophage, allows the viral DNA to bind selectively to transcription factor 1. We have synthesized a TF1-binding site: d(5'-ACCHACHCHHHGHAGGT-3')-d(5'-ACCHACAAAGAGHAGGT-3') and studied this molecule using NMR spectroscopy. The chemical shifts of exchangeable and non-exchangeable protons were sequentially assigned. Absence of corresponding NOEs in the imino-imino region suggested that the end base pairs did not form Watson-Crick hydrogen bond. Restrained molecular dynamics calculation yielded a family of B-DNA structures whose r.m.s.d. was 0.66 A (all atoms) for the internal 15 bp. The helical twist was 38.5 degrees per step. The base pairs were situated directly on the helix axis (X-displacement = -0.2 A). All sugars exhibited C2'-endo puckering with P = 167.3 degrees and upsilon(max)= 38.2 degrees. The OH groups of all hmU bases resided on the 3' side of the base plane and may affect the base orientation relative to the sugar plane as the average chi value for all hmU was 4 degrees more positive than that of other nucleosides (258 degrees versus 254 degrees ). Positive roll angles (rho) and small flanking twists (omega) at hmU suggested that the two hmU-A base pair steps open toward the minor grooves.

[Nonvaccine Streptococcus pneumoniae serotypes causing acute bacterial meningitis]. / Streptococcus pneumoniae serotipo no vacunal como agente causal de meningitis bacteriana aguda.

The pneumococcal heptavalent conjugate vaccine protects children aged less than 2 years old from invasive pneumococcal disease (IPD). Efficacy is 89-93% in the US population and 71-86% in European studies. The vaccine confers active immunization against the main serotypes causing IPD (4, 6B, 9V, 14, 18C, 19F y 23F). We describe 2 children who presented with pneumococcal meningitis caused by nonvaccine serotypes. As a result of the widespread use of the heptavalent vaccine, there may be a shift in the serotypes causing IPD.

Different bindings of the minor groove ligands DAPI and Hoechst 33258 to multimers of the curved (CA4T4G) and noncurved (CT4A4G) DNA sequences.

Equilibrium binding properties of DAPI and Hoechst for prototype curved (CA4T4G)n and straight (CT4A4G)n DNAs were derived from fluorescence intensity. Both decamers display a single strong binding site for each ligand. Hoechst binds seven times stronger to curved than to straight DNA. An additional cooperative, much weaker binding site is found for DAPI.

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.

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.

Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SPO1-encoded type II DNA-binding protein TF1.

TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.

Synthesis of two distamycin analogues and their binding mode to d(CGCAAATTTGCG)2 in the 2:1 solution complexes as determined by two-dimensional 1H-NMR.

In the course of a study aimed at the synthesis of pyrrole amidine carboxamide DNA-binding agents as novel pharmacological agents, a series of carbamoyl analogues of distamycin, containing an increasing number of pyrrole units, have been obtained by total synthesis. The interaction of the tetrapyrrole carbamoyl 4 with the dodecamer d(CGCAAATTTGCG)2 in comparison with that of the corresponding formylamino analogue 3 has been examined by high-resolution 1H-NMR and molecular modeling. Either ligand binds to DNA in one-drug and symmetric two-drug modes at low drug:DNA ratios, while at high ratios only the two-drug complex was observed. In this article, the structure of 2:1 drugs DNA complexes has been studied by NMR and molecular modeling, which indicate that the two analogues bind the DNA in a similar fashion, in the minor groove of the 5'-AATTT region. In both complexes the two drugs are symmetrically placed along the complementary strands of DNA with the pyrrole ring of one molecule in close contact with those of the other one. Although another region of five consecutive A-T base pairs is available, no evidence of sliding of drug molecules between different binding sites, as in the case of the 2:1 complex of distamycin with the same dodecamer, is observed, thus indicating that increasing the number of N-methylpyrrolecarboxamide units from three to four cases a lengthening of the recognition sequence.

An NMR study of the conformation and thermodynamics of the circular dumbbell d [formula: see text] Slow exchange between two- and four-membered hairpin loops.

The circular DNA decamer 5'-d [formula: see text] 3' is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5'-GTTC-3' loop is disrupted, whereas the opposite 5'-CTTG-3' loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.

Once-weekly epoetin alfa for treating the anemia of chronic kidney disease.

BACKGROUND AND AIM: Anemia occurs in approximately 47% of patients with chronic kidney disease (CKD) not on dialysis. Recombinant human erythropoietin (r-HuEPO, epoetin alfa) has been proven safe and effective for anemia treatment in patients with CKD using a three times-weekly regimen. The current study was conducted to evaluate the clinical safety and efficacy of a less frequent dosing regimen (once weekly) in this population. METHODS: This prospective, multicenter, open-label, non-randomized study enrolled 1,557 adult anemic (hemoglobin (Hb) < or = 10 g/dl) CKD patients not on dialysis. Epoetin alfa 10,000 U was administered subcutaneously once weekly for 16 weeks. Titration to 20,000 U once weekly at week 5 was permitted if patients had an increase in Hb < 1 g/dl. Safety and efficacy were assessed by changes in health-related quality of life (Linear Analog Scale Assessment (LASA) and Kidney Disease Questionnaire (KDQ)), changes in hematologic parameters and transfusion utilization, and incidence and severity of adverse events. RESULTS: 1,338 patients were evaluable for efficacy. Mean Hb level increased from 9.1 g/dl at baseline to 11.6 g/dl at study completion (last observed value after baseline) (p < 0.0001). Overall, 89.8% of patients responded to once-weekly dosing, exhibiting an increase in Hb level of > or = 1 g/dl from baseline. The percentage of patients that required transfusion decreased from 11.1% (baseline) to 3.7% (during the study) (p < 0.0001). All quality-of-life parameters improved significantly from baseline (p < 0.0001). Mean LASA scores for energy, activity and overall quality of life increased from baseline to study completion by 27.9 mm (70.5%), 24.5 mm (57.0%) and 22.6 mm (47.4%), respectively. All 5 KDQ domains showed statistically significant improvements (p < 0.0001). Hb change was a strong predictor for all 5 KDQ domains and the overall score (p < 0.0001). Treatment with once-weekly epoetin alfa was well tolerated, similar to that reported with three times-weekly dosing. CONCLUSION: Once-weekly epoetin alfa therapy is safe and effective for treating anemia in patients with CKD not on dialysis, and is associated with significant improvements in functional status and quality of life.

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.

Effect of hyaluronic acid amide derivative on equine synovial fluid viscoelasticity.

An amphiphilic hyaluronic acid (HA) derivative has been obtained by the amidation of the carboxylic group of the glucuronic acid. This derivative, HYADD4-G (HY4), is the hexadecylamide of 500-730 kDa hyaluronic acid, derived from Streptococcus equi at about 2% degree of substitution (2 mol hexadecylamine per 100 mol hexuronic acid). Its viscoelastic properties, at a concentration of 5 mg/mL in phosphate buffer saline, have been compared with those solutions of native HA, having the same molecular weight. Changes in the viscoelastic properties of equine synovial fluid (SF) when mixed with HY4 over a series of volume ratios-viz 1:2, 1:1, 3:1, and 7:1-have been evaluated. HY4 is able to associate into aqueous solution, and its rheological behavior is typical of a weak gel. Throughout the frequency range investigated (0.1-10 Hz), the elastic modulus G' is higher than the viscous modulus G'', and both moduli are frequency independent, and G' value is about two orders of magnitude higher than that of a comparable solution of native HA. The addition of HY4 to equine synovial fluid (SF) increased its viscoelasticity at all the SF:HY4 ratios tested. These results demonstrate that HY4 is able to integrate with SF, increasing the synovial fluid rheology, and could be an interesting new option in viscosupplement therapy of osteoarthritis, particularly considering its low degree of chemical modification from native HA.

Effects of fibronectin and laminin on structural, mechanical and transport properties of 3D collageneous network.

Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN- and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN- and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.