Enrico Bucci

Selection of D-Amino-Acid peptides that bind to Alzheimer's disease amyloid peptide A beta(1-42) by mirror image phage display

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimers disease amyloid peptide Aβ(1–42). A randomized 12‐mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Aβ(1–42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D‐pep) was shown to bind Aβ(1–42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimers disease, amyloid plaques and leptomeningeal vessels containing Aβ amyloid were stained specifically with a fluorescence‐labeled derivative of D‐pep. Fibrillar deposits derived from other amyloidosis were not labeled by D‐pep. Possible applications of this novel and highly specific Aβ ligand in diagnosis and therapy of Alzheimers disease are discussed.

Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C.

The initiation of enteroviral positive-strand RNA synthesis requires the presence of a functional ribonucleoprotein complex containing a cloverleaf-like RNA secondary structure at the 5 end of the viral genome. Other components of the ribonucleoprotein complex are the viral 3CD proteinase (the precursor protein of the 3C proteinase and the 3D polymerase), the viral 3AB protein and the cellular poly(rC)-binding protein 2. For a molecular characterization of the RNA-binding properties of the enteroviral proteinase, the 3C proteinase of coxsackievirus B3 (CVB3) was bacterially expressed and purified. The recombinant protein is proteolytically active and forms a stable complex with in vitro-transcribed cloverleaf RNA of CVB3. The formation of stable complexes is also demonstrated with cloverleaf RNA of poliovirus (PV) 1, the first cloverleaf of bovine enterovirus (BEV) 1, and human rhinovirus (HRV) 2 but not with cloverleaf RNA of HRV14 and the second cloverleaf of BEV1. The apparent dissociation constants of the protein:RNA complexes range from approx. 1.7 to 4.6 microM. An electrophoretic mobility shift assay with subdomain D of the CVB3 cloverleaf demonstrates that this RNA is sufficient to bind the CVB3 3C proteinase. Binding assays using mutated versions of CVB3 and HRV14 cloverleaf RNAs suggest that the presence of structural features rather than a defined sequence motif of loop D are important for 3C proteinase-RNA interaction.

NMR Structure of the Single QALGGH Zinc Finger Domain from the Arabidopsis thaliana SUPERMAN Protein

Zinc finger domains of the classical type represent the most abundant DNA binding domains in eukaryotic transcription factors. Plant proteins contain from one to four zinc finger domains, which are characterized by high conservation of the sequence QALGGH, shown to be critical for DNA‐binding activity. The Arabidopsis thaliana SUPERMAN protein, which contains a single QALGGH zinc finger, is necessary for proper spatial development of reproductive floral tissues and has been shown to specifically bind to DNA. Here, we report the synthesis and UV and NMR spectroscopic structural characterization of a 37 amino acid SUPERMAN region complexed to a Zn2+ ion (Zn–SUP37) and present the first high‐resolution structure of a classical zinc finger domain from a plant protein. The NMR structure of the SUPERMAN zinc finger domain consists of a very well‐defined ββα motif, typical of all other Cys2‐His2 zinc fingers structurally characterized. As a consequence, the highly conserved QALGGH sequence is located at the N terminus of the α helix. This region of the domain of animal zinc finger proteins consists of hypervariable residues that are responsible for recognizing the DNA bases. Therefore, we propose a peculiar DNA recognition code for the QALGGH zinc finger domain that includes all or some of the amino acid residues at positions −1, 2, and 3 (numbered relative to the N terminus of the helix) and possibly others at the C‐terminal end of the recognition helix. This study further confirms that the zinc finger domain, though very simple, is an extremely versatile DNA binding motif.

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‐ε‐peptide that we called ε‐lysPNA. The ability to bind complementary RNA and the stability in serum of this synthetic nucleo‐ε‐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 ε‐peptides and other ε‐amino acid‐containing compounds in natural products with biological activity such as the poly‐ε‐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 ε‐peptides, and not α‐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 ε‐lysPNAs encourage, without doubts, interest in the therapeutic and diagnostic potential of these bioinspired nucleopeptides. Copyright

Computational Procedures to Explain the Different Biological Activity of DNA/DNA, DNA/PNA and PNA/PNA Hybrid Molecules Mimicking NF-κB Binding Sites

Abstract Peptide nucleic acids (PNA) have recently been proposed as alternative reagents in experiments aimed to the control of gene expression. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units and therefore is stable in human serum and cellular extracts. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and giving rise to highly stable (PNA)2-RNA triplexes with RNA targets. Therefore, antisense and antigéne PNAs have been synthetized and characterized. The major issue of the present paper is to describe some computational procedures useful to compare the behaviour of PNA double stranded molecules and PNA/DNA hybrids with the behaviour of regular DNA duplexes in generating complexes with DNA-binding proteins. The performed computational analyses clearly allow to predict that the lack of charged phosphate groups and the different shape of helix play a critical role in the binding efficiency of NF-κB transcription factors. These computational analyses are in agreement with competitive gel shift and UV-cross linking experiments. These experiments demonstrate that NF-κB PNA/PNA hybrids do not interact efficiently with proteins recognizing the NF-KB binding sites in genomic sequences. In addition, the data obtained indicate that the same NF-κB binding proteins recognize both the NF-κB DNA/PNA and DNA/DNA hybrids, but the molecular complexes generated with NF-κB DNA/PNA hybrids are less stable than those generated with NF-κB target DNA/DNA molecules.

DNA-based strategies for blocking HMGB1 cytokine activity: design, synthesis and preliminary in vitro/in vivo assays of DNA and DNA-like duplexes

In this work we report the design and synthesis of kinked oligonucleotide duplexes as potential inhibitors of HMGB1, a cytokine which triggers a broad range of immunological effects. We found that the designed ligands can interact with HMGB1, as evidenced by circular dichroism spectroscopy, and are able to block some extracellular effects induced by the protein, such as cellular proliferation and migration, as we demonstrated by in vitro biological assays. After selecting the most stable and active kinked duplex, we synthesized the corresponding PNA/DNA chimeric duplex which resulted to be more resistant to enzymatic degradation, and showed a biological activity comparable to that of the natural duplex. Preliminary in vivo assays in a mouse inflammatory model, showed a significant decrease of the mortality after administration of the PNA/DNA kinked duplex to LPS-treated mice.

Evidences of complex formation between DABA-based nucleo-γ-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‐NH2, H‐G‐(t D−dab)6‐K‐NH2, H‐G‐(a L−dab)6‐K‐NH2, H‐G‐(t L−dab‐t D−dab)3‐K‐NH2, H‐G‐(a L−dab‐a D−dab)3‐K‐NH2, H‐G‐(a L−dab‐t D−dab)3‐K‐NH2. 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. Copyright