Anti-HIV activity of new higher order G-quadruplex aptamers obtained from tetra-end-linked oligonucleotides.

By combining the ability of short G-rich oligodeoxyribonucleotides (ODNs) containing the sequence 5'CGGA3' to form higher order G-quadruplex (G4) complexes with the tetra-end-linked (TEL) concept to produce aptamers targeting the HIV envelope glycoprotein 120 (gp120), three new TEL-ODNs (1-3) having the sequence 5'CGGAGG3' were synthesized with the aim of studying the effect of G4 dimerization on their anti-HIV activity. Furthermore, in order to investigate the effect of the groups at the 5' position, the 5' ends of 1-3 were left uncapped (1) or capped with either the lipophilic dimethoxytrityl (DMT) (2) or the hydrophilic glucosyl-4-phosphate (3) moieties. The here reported results demonstrate that only the DMT-substituted TEL-ODN 2 is effective in protecting human MT-4 cell cultures from HIV infection (76% max protection), notwithstanding all the three new aptamers proved to be capable of forming stable higher order dimeric G4s when annealed in K+-containing buffer, thus suggesting that the recognition of a hydrophobic pocket on the target glycoprotein by the aptamers represents a main structural feature for triggering their anti-HIV activity.

Solid phase synthesis and RNA-binding activity of an arginine-containing nucleopeptide.

Here we report the solid phase synthesis and characterization (LC-ESIMS, CD) of a cationic nucleobase-containing α-peptide, composed of both l-arginine residues and l-lysine-based nucleoamino acids sequentially present in the structure. The binding properties of this novel basic nucleopeptide towards nucleic acids were investigated by CD spectroscopy which revealed the ability of the thymine-containing oligomer to bind both adenine-containing DNA (dA12) and RNA (poly rA) molecules inducing high conformational variations in the nucleic acid structures. Moreover, the artificial oligonucleotide inhibited the enzymatic activity of HIV reverse transcriptase, opening the door to the exploitation of novel antiviral strategies inspired to this molecular tool.

Synthesis, characterization and hybridization studies of an alternate nucleo-epsilon/gamma-peptide: complexes formation with natural nucleic acids.

In order to develop new oligodeoxyribonucleotide (ODN) analogs to be used in biotechnological applications, we report here the synthesis, characterization and nucleic acid binding studies of novel nucleopeptides, that we called epsilon-lys/gamma-dabPNAs, containing a backbone of alternated L-diaminobutyric acid and L-lysine moieties. Exploring the hybridization properties of the new ODN analog, we found, by circular dichroism and UV spectroscopies, that a homothymine epsilon-lys/gamma-dabPNA hexamer binds both DNA and RNA of complementary sequence. Furthermore, human serum stability assays on the alternate nucleopeptide evidenced a noteworthy degradation resistance. These results encourage us to deepen the knowledge of this analog, in order to evaluate its possible use in antigene/antisense or diagnostic applications.

Solid phase synthesis and RNA-binding studies of a serum-resistant nucleo-epsilon-peptide.

In the present work we report the synthesis of a new Fmoc-protected L-lysine-based nucleo-amino acid suitable for the solid phase assembly and its oligomerisation to the corresponding nucleo-epsilon-peptide that we called epsilon-lysPNA. The ability to bind complementary RNA and the stability in serum of this synthetic nucleo-epsilon-peptide were studied to explore its possible use in antisense or diagnostic applications. Our interest to the presented oligonucleotide analogue was also supported by the importance of epsilon-peptides and other epsilon-amino acid-containing compounds in natural products with biological activity such as the poly-epsilon-lysines produced by Streptomyces albulus that possess a highly selective antimicrobial activity. Another aspect we intended to evaluate by this work is the possible prebiotic implication of these nucleopeptides, since epsilon-peptides, and not alpha-peptides, were mainly obtained among the other thermal prebiotic polypeptides in pyrocondensation of lysine, a diamino acid also detected in Mighei meteorite. Besides this intriguing question, all the remarkable properties emerged from the present investigation on epsilon-lysPNAs encourage, without doubts, interest in the therapeutic and diagnostic potential of these bioinspired nucleopeptides.

Evidences of complex formation between DABA-based nucleo-gamma-peptides with alternate configuration backbone.

In the present work, we report the synthesis and the characterization of dab PNA hexamers with diaminobutyric acid backbone of D- or/and L-configuration. In particular, the four nucleo-amino acids we synthesized, D- and L-diaminobutyryl adenines and D- and L-diaminobutyryl thymines, were used in various combinations to assemble the following oligomers: H-G-(t( L-dab))(6)-K-NH(2), H-G-(t( D-dab))(6)-K-NH(2), H-G-(a( L-dab))(6)-K-NH(2), H-G-(t( L-dab)-t( D-dab))(3)-K-NH(2), H-G-(a( L-dab)-a( D-dab))(3)-K-NH(2), H-G-(a( L-dab)-t( D-dab))(3)-K-NH(2). By using CD and UV spectroscopies, we investigated the ability of complementary dab PNA strands to bind to each other. We found that binding occurs only between oligomers with backbone of alternate configuration [(t( L-dab)-t( D-dab))(3)/(a( L-dab)-a( D-dab))(3) and (a( L-dab)-t( D-dab))(3)/(a( L-dab)-t( D-dab))(3)] and implies cooperative hydrogen bonds and base stacking. Furthermore, interesting properties relative to the self-complementary oligomer (a( L-dab)-t( D-dab))(3) forming palindromic complexes emerged from preliminary dynamic light-scattering experiments that suggested the formation of multimeric aggregates. These results, together with the high serum stability of the DABA-based oligomers, as shown by HPLC analysis, encourage us to further study dab PNAs as new self-recognizing bio-inspired polymers, to develop new nanomaterials in biotechnological and biomedical applications.

Bent oligonucleotide duplexes as HMGB1 inhibitors: a comparative study.

In this work we explore the ability of a chimeric LNA/DNA bent duplex, in which the kink is induced by 2 unpaired adenines in the middle of one strand, to bind HMGB1, a protein involved in many inflammatory processes. The LNA/DNA duplex was compared with the corresponding full DNA and PNA/DNA chimera duplexes from a thermodynamic and spectroscopic point of view.

ODN-based drugs for targeting of extracellular proteins.

In this work a novel approach to identify new therapeutic targets consisting of serum proteins which contain an oligonucleotide binding domain is presented.

Synthesis, characterization and hybridization studies of new nucleo-gamma-peptides based on diaminobutyric acid.

In the present work, we report the synthesis and the characterization of a new chiral nucleoaminoacid, in which a diaminobutyric moiety is connected to the DNA nucleobase by an amidic bond, and its oligomerization to give the corresponding nucleo-gamma-peptide. The ability of this synthetic polymer to bind complementary DNA was studied in order to explore its possible use in antigene/antisense or diagnostic applications. Our interest in the presented DNA analogue was also supported by the importance of gamma-aminoacid-containing compounds in natural products of biological activity and by the known stability of gamma-peptides to enzymatic degradation. Furthermore, our work could contribute to the study of the role of nucleopeptides as prebiotic material in a PNA world that could successively lead to the actual DNA/RNA/protein world, as recently assumed.

Binocular field in pigeons: behavioral measures of stimulus detection and coding.

The extension of the binocular visual field of pigeons is measured using a pecking behavioral method. Our estimate of the binocular field derived at the animal near point of accommodation indicates a 50 degrees width on the horizontal axis and more than 50 degrees on the vertical axis. Within the binocular field there is a core of about 24 degrees, named coding area, in which the stimulus is detected and coded by only one head fixation before accomplishing the pecking response, in both binocular and monocular viewing conditions. Outside the coding area, lies the detection area, in which the stimulus can be detected, but it needs a second head fixation to be coded in all viewing conditions. Our results are in agreement with the hypothesis that the coding area corresponds to the red field projection of each retina. In binocular vision an overlap of the two areas occurs, thus promoting the image fusion useful for a stereoscopic vision during the pecking to the seed.

Binocular probability summation in a choice reaction-time task in pigeons.

We present evidence for binocular summation in pigeons in a choice reaction time paradigm. Considering binocular vision as the result of a race between the equivalent eyes, pigeons can adopt both a 'subject by subject' and a 'trial by trial' race model, and binocular advantage can be attributed merely to a probability summation. Pigeons were tested on a visual discrimination task in which the time for detection was gradually reduced and a quick response was required. The time presentation of a small black spot could vary from 2000 ms to the shortest value (157-226 ms) allowing maintenance of a satisfactory performance criterion.

Interocular transfer of depth discrimination in intact and in DSO-sectioned pigeons.

Interocular transfer (IOT) of a depth discrimination task was studied in intact pigeons and with a section of the supraoptic decussation (DSO). Animals were trained to respond to the nearer of two small light emitting diodes placed at different depths in the left and right compartments of a black tunnel. The near stimulus (at 10.5 cm from the eyes) and the far one (at 21 cm) could only be seen one at a time. Though the task was difficult to learn monocularly, intact as well as lesioned animals had good transfer scores with the untrained eye. Success in transfer may be related to the presentation of the discriminanda which assured that their images impinged upon the retinal 'red field'. DSO-transection did not affect IOT possibly because differential oculomotor adjustments needed for focusing near or far targets provide central bilateral and/or binocular information which is not conveyed by the DSO.

Binocular interactions measured by choice reaction times in pigeons.

In 7 pigeons binocular interactions were studied by recording monocular and binocular choice reaction times (RTs) to a pattern discrimination task. In all the animals binocular responses were found to be significantly faster than monocular RTs. In order to assess whether binocular advantage might be ascribed to a neural interaction between the two eyes, monocular and binocular performances were evaluated according to a probability summation model. The results indicate that in the great majority of animals, probability summation alone can account for binocular superiority.

An automated apparatus for the study of visual discriminations in pigeons.

A fully automated apparatus for the study of visual discriminations in pigeons is described. The apparatus allows the presentation of a single stimulus for successive discriminations and of two or more stimuli at the same time for simultaneous discriminations. It has proven advantageous for comparing successive and simultaneous discriminations in tests of interocular transfer.

Interocular transfer of visual discriminations in Wulst-ablated pigeons.

Interocular transfer (IOT) of monocularly learned pattern, brightness and color discriminations was studied in normal pigeons and in pigeons with a bilateral ablation of the visual Wulst. No significant differences were found between the two groups in terms of both rate of monocular learning and degree of IOT. Pigeons with a Wulst ablation which had shown a normal capacity for IOT, subsequently lost this capacity following a section of the supra-optic decussation (DSO). It is concluded that IOT in pigeons does not require the presence of the binocular neurons of the visual Wulst, and that in absence of this structure, IOT must rely on other centers for binocular interaction which receive fibers from the DSO.

Apomorphine pecking in the pigeon.

Pecking activity elicited by apomorphine was studied quantitatively in intact and thalamic pigeons. While apomorphine pecking is easily observed in the intact pigeon, it is absent in the acute thalamic animal. I reappears, however, in the chronic preparation simultaneously with the recovery of spontaneous pecking. Apomorphine pecking is described by satiation and increased by fasting. Reticular stimulation produces reversible blockade of both apomorphine and spontaneous pecking with the same parameters, without increase in heart rate. Apomorphine pecking can be observed only in the presence of visual contrast, and is always aimed at the contrast points or edges. A decrease in contrast produces a decrease in the number of pecks delivered to the contrast points and an increase in those missing the target, while the total number of pecks delivered in a unit time is unaffected. A lowering of the background luminance is followed by a decrease in the total number of pecks, which is mostly due to a reduction of those aimed at the contrast points. All these findings are discussed and it is concluded that: i) apomorphine exerts an excitatory action (direct or indirect) on the hypothalamic feeding centers; hence apomorphine pecking can be considered as a pharmacologically motivated behavior; ii) visual contrast exerts a dual action on apomorphine pecking, namely it acts both as triggering stimulus and goal object; iii) the decrease in apomorphine pecking induced by lowering the ambient light intensity is probably due to a decrease in the level of the general arousal.

Apparatus for quantitative evaluation of visually guided pecking in the pigeon.

An apparatus using a phase-locked loop is described which allows automated, differential counts of correct and incorrectly oriented pecks, elicited by apomorphine in the pigeon. This apparatus provides a unique and precise means for the study of this characteristic, visually guided behavior.