A non-covalent peptide-based carrier for in vivo delivery of DNA mimics.

The dramatic acceleration in identification of new nucleic-acid-based therapeutic molecules has provided new perspectives in pharmaceutical research. However, their development is limited by their poor cellular uptake and inefficient trafficking. Here we describe a short amphipathic peptide, Pep-3, that combines a tryptophan/phenylalanine domain with a lysine/arginine-rich hydrophilic motif. Pep-3 forms stable nano-size complexes with peptide-nucleic acid analogues and promotes their efficient delivery into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. We demonstrate that Pep-3-mediated delivery of antisense-cyclin B1-charged-PNA blocks tumour growth in vivo upon intratumoral and intravenous injection. Moreover, we show that PEGylation of Pep-3 significantly improves complex stability in vivo and consequently the efficiency of antisense cyclin B1 administered intravenously. Given the biological characteristics of these vectors, we believe that peptide-based delivery technologies hold a true promise for therapeutic applications of DNA mimics.

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling.

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.

Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics.

Negatively charged homo-oligomers of alternating trans-4-hydroxy-L-proline/phosphonate polyamides with DNA bases (HypNA-pPNA) display excellent hybridization properties toward DNA and RNA, while preserving the mismatch discrimination, nuclease resistance, and protease resistance of peptide nucleic acids (PNAs). Similar properties are associated with morpholino phosphorodiamidate (MO) DNA mimics, which have been used in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. We evaluated mixed sequence HypNA-pPNAs as an alternative to MOs, and found that even a single central DNA mismatch lowered the HypNA-pPNA melting temperature by 16 degrees C. We then observed that the melting temperatures of HypNA-pPNA 18-mers hybridized to RNA 25-mers were comparable to the melting temperatures of MO 25-mers, and that two HypNA-pPNA mismatches lowered the melting temperature with RNA by 18 degrees C. In zebrafish embryos we observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail, and uroD, with greater mismatch stringency. Finally we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. HypNA-pPNAs designed to inhibit translation of specific zebrafish RNA targets thus demonstrated stringent hybridization properties, relative to DNA and MO oligomers, and present a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes.

Downregulation of gene expression with negatively charged peptide nucleic acids (PNAs) in zebrafish embryos.

We found that negatively charged, highly soluble PNA analogs with alternating phosphonates (HypNA-pPNAs) are effective and specific antisense agents in zebrafish embryos, showing comparable potency and greater specificity against chordin, ntl and uroD. In addition, we successfully phenocopied a dharma mutant that had not been found susceptible to MO knockdown. Both MO and HypNA-pPNAs against a tumor suppressor gene induced comparable upregulation of p53, illustrating similar effects on transcription profiles. HypNA-pPNAs are therefore a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes in zebrafish or validating newly identified orthologs, and perhaps for reverse genetic studies in other organisms.