Artificial DNA: PNA & XNA最新文献

We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept)avidin-coated fluorescent beads, differing in size and color [green beads (1 µm) and red beads (5.9 µm)], thereby allowing distinct detection of each PNA probe by conventional fluorescence microscopy. These two PNA beads showed easily detectable co-localization when simultaneously hybridizing to a target nucleic acid. The assay detected the parasite 18S rRNA down to 1.6 fmol while there was no such co-localization visible with human 18S rRNA not containing the PNA targets. Furthermore, the assay showed positive detection with 1.6 ng of total RNA (corresponding to RNA from ca. 300 parasites). Upon further optimization this method may provide a new tool for a diagnosis of Human African Trypanosomiasis (HAT) and it may more generally have applications within diagnostics for (neglected) infectious diseases.

Pseudo-complementary oligonucleotide analogues and mimics provide novel opportunities for targeting duplex structures in RNA and DNA. Previously, a pseudo-complementary A-T base pair has been introduced. Towards sequence unrestricted targeting, a pseudo-complementary G-C base pair consisting of the unnatural nucleobases n6-methoxy-2,6-diaminopurine (previously described in a DNA context) and N4-benzoylcytosine is now presented for design of pseudo-complementary PNA oligomers (pcPNAs).

A recent claim is discussed that Watson-Crick pairs in the naked duplex DNA spontaneously flip into Hoogsteen pairs under ordinary conditions. The claim is considered within the historical retrospective and is put into the broader context of DNA biophysics.

The recent paper by Wolfe-Simon et al.1 reporting a bacterial strain, which is able to grow in high concentrations of arsenate, apparently in the absence of phosphate, and claims that in this strain arsenate is substituting for phosphate, e.g. in nucleic acids (Figure 1), was highly profiled, attracted broad attention, and almost immediately resulted in heavy scientific criticism (see e.g. 2-7).

Molecular computing is potentially one of the most powerful tools for the development of massive parallel computing protocols. In the present paper, a first example of the use of PNA:PNA interactions in molecular computing is described. A series of short PNA sequences have been designed with a four base stretch coding for variables and solutions. Hybridization of the components in different combinations was tested both in solution and in a microarray format. A series of PNA representing the solutions were spotted on a microarray surface in order to simulate the hardware. A series of PNA representing the variables, labeled with TAMRA, were used to interrogate the device enabling to solve non-deterministic logic operations. The system was shown to be able to solve a two-variable equation with a high signal to noise ratio. This paper intends to provide a proof of principle that PNA, on account of their stability and specificity of binding, are most suitable for constructing organic-type computers.

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA).

Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies.

The University of Texas researchers have recently discovered that small synthetic RNAs (sRNAs) that are complementary to sequences located 3'-outside of genes can efficiently modulate gene expression. These new findings significantly expand the transcription-regulatory potential of sRNAs, and they also may provide useful leads for other artificial nucleobase oligomers to target sequences beyond the 3' termini of mRNA.

1,2,3-triazole analogues of PNA (TzNA) in which the amide link in backbone is replaced by triazole ring is synthesized on solid phase by 'click' chemistry and such triazolothymine PNA chimeric oligomers are shown to significantly stabilize the derived PNA(2):DNA triplexes. With increasing number of triazole units in the backbone, single stranded PNA oligomers exhibit enhanced self-ordering.

The design and the synthesis of a PNA oligomer containing a pyrenyl residue in the backbone were performed. PNA sequence was chosen complementary to a "G rich" target sequence involved in G-quadruplex formation. The pyrenyl unit replaced a nucleobase in the middle of the PNA through covalent linkage to the backbone by a carboxymethyl unit. A systematic study on the binding properties of this probe towards DNA and RNA complementary strands was carried out by UV and fluorescence spectroscopy. UV melting curves indicated that the PNA probe binds more tightly to RNA rather than to DNA. Thermodynamic data obtained by Van't Hoff fitting of the melting curves indicated that, in the case of RNA, a more favorable interaction occurs between the pyrenyl unit and the RNA nucleobases, leading to a very favorable enthalpic contribution.The fluorescence analysis showed specific quenching of the pyrene emission associated to the formation of the full-match PNA-DNA or PNA-RNA duplexes. Again, this behavior was more evident in the case of RNA, consistently with the stronger interaction of the pyrenyl unit with the complementary strand. In order to study the sequence specificity of the pyrenyl-PNA probe (pyr-PNA), recognition experiments on mismatched DNA and RNA sequences were also performed.