Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

The Solution Synthesis of Antisense Oligonucleotide‐Peptide Conjugates Directly Linked via Phosphoramide Bond by Using a Fragment Coupling Approach

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell‐penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1‐hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2′‐dithiopyridine and 4‐dimethylaminopyridine in organic media. Several oligonucleotides, including a 18‐mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.     

E. COLI RNASE HI AND THE PHOSPHONATE-DNA/RNA HYBRID: MOLECULAR DYNAMICS SIMULATIONS

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3′–O–CH₂–P–O–5′ or 3′–O–P–CH₂–O–5′) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3′-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg⁺⁺ · 4H₂O chelate complex (bound in the active site) were analyzed in detail. Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Synthesis and Application of Negatively Charged PNA Analogues

Negatively charged DNA mimics containing phosphonate analogues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.     

Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.