Artificial DNA: PNA & XNA最新文献

PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs.

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.

One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3-1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.

Peptide nucleic acid (PNA) is one of the most widely used synthetic DNA analogs. Conjugation of functional molecules to PNA is very effective to further widen its potential applications. For this purpose, here we report the synthesis of several ligand monomers and introduced them to PNA. These ligand-modified PNAs attract cerium ion and are useful for site-selective DNA hydrolysis. It should be noted that these ligands on PNA are also effective even under the conditions of invasion complex.

The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present evidence that the identification of arsenate DNA was artifactual.

A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.

Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.

The design and facile synthesis of sterically constrained new analogs of PNA having gem-dimethyl substitutions on glycine (dmg-PNA-T) is presented. The PNA oligomers [aminoethyl dimethylglycyl (aedmg) and aminopropyl dimethylglycyl (apdmg)] synthesized from the monomers 6 and 12) effected remarkable stabilization of homothyminePNA(2):homoadenine DNA/RNA triplexes and mixed base sequence duplexes with target cDNA or RNA. They show a higher binding to DNA relative to that with isosequential RNA. This may be a structural consequence of the sterically rigid gem-dimethyl group, imposing a pre-organized conformation favorable for complex formation with cDNA. The results complement our previous work that had demonstrated that cyclohexanyl-PNAs favor binding with cRNA compared with cDNA and imply that the biophysical and structural properties of PNAs can be directed by introduction of the right rigidity in PNA backbone devoid of chirality. This approach of tweaking selectivity in binding of PNA constructs by installing gem-dimethyl substitution in PNA backbone can be extended to further fine-tuning by similar substitution in the aminoethyl segment as well either individually or in conjunction with present substitution.