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.     

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.     

Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro

Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.     

RNAi and microRNA: breakthrough technologies for the improvement of plant nutritional value and metabolic engineering

This article raises the complex issue of improving plant nutritional value through metabolic engineering and the potential of using RNAi and micro RNA technologies to overcome this complexity, focusing on a few key examples. It also highlights current knowledge of RNAi and microRNA functions and discusses recent progress in the development of new RNAi vectors and their applications. RNA interference (RNAi) and microRNA (miRNA) are recent breakthrough discoveries in the life sciences recognized by the 2006 Nobel Prize in Physiology or Medicine. The importance of these discoveries relates not only to elucidating the fundamental regulatory aspects of gene expression, but also to the tremendous potential of their applications in plants and animals. Here, we review recent applications of RNAi and microRNA for improving the nutritional value of plants, discuss applications of metabolomics technologies in genetic engineering, and provide an update on the related RNAi and microRNA technologies.     

A functionalized 20-residue peptaibol derivative for nucleic acid delivery

Based on the membrane-modifying peptaibol trichocellin-A-I (1) from Trichoderma viride, we designed a vehicle for the cellular delivery of antisense oligodeoxynucleotides by attaching a (Lys)10 stretch to the C-terminus of 1. The resulting transporter peptide 2, prepared by solid-phase synthesis using Fmoc protocol in combination with amino acid fluorides, was found to be mainly alpha-helical in solution, in contrast to its precursors 1 and 3. The uptake of the complex formed between carrier 2 and a fluorescence-tagged oligonucleotide, i.e., 4, was studied at different charge ratios by confocal laser-scanning microscopy, using two different eukaryotic cell lines: mouse embryonal fibroblast (NIH3T3) and human lung carcinoma (A549) cells. Peptide 2 readily translocated 4 into the cytoplasms of NIH3T3 cells. However, the peptide/oligonucleotide complex was accumulated around the plasma membrane of the A549 cells.     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.