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26.  Peptide nucleic acids tagged with four lysine residues for amperometric genosensors 
Artificial DNA, PNA & XNA  2012;3(2):80-87.
A homothymine PNA decamer bearing four lysine residues has been synthesized as a probe for the development of amperometric sensors. On one hand, the four amino groups introduced make this derivative nine times more soluble than the corresponding homothymine PNA decamer and, on the other hand, allow the stable anchoring of this molecule on Au nanostructured surface through the terminal -NH2 moieties. In particular, XPS and electrochemical investigations performed with hexylamine, as a model molecule, indicate that the stable deposition of primary amine derivatives on such a nanostructured surface is possible and involves the free electron doublet on the nitrogen atom. This finding indicates that this PNA derivative is suitable to act as the probe molecule for the development of amperometric sensors.
 
Thanks to the molecular probe chosen and to the use of a nanostructured surface as the substrate for the sensor assembly, the device proposed makes possible the selective recognition of the target oligonucleotide sequence with very high sensitivity.
doi:10.4161/adna.20777
PMCID: PMC3429534  PMID: 22772036
DNA recognition; PNA; amperometric genosensors; nanostructured surfaces; amine deposition
27.  Targeting pre-miRNA by Peptide Nucleic Acids 
Artificial DNA, PNA & XNA  2012;3(2):88-96.
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.
doi:10.4161/adna.20911
PMCID: PMC3429535  PMID: 22699795
FACS; fluorescence; miR-210; PNA; pre-miR; thiazole orange
28.  Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3–1 cells 
Artificial DNA, PNA & XNA  2012;3(2):97-296.
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.
doi:10.4161/adna.21061
PMCID: PMC3429536  PMID: 22772035
NF-kappaB; transcription factor decoy; inflammation; Peptide Nucleic Acids; PNA-DNA chimeras
29.  Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides 
Artificial DNA, PNA & XNA  2012;3(1):14-21.
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5′-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°Nm, Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.
doi:10.4161/adna.19272
PMCID: PMC3368812  PMID: 22679529
enzymatic synthesis; modified nucleotide; polymerase; triphosphate; α-L-LNA
30.  Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods 
Artificial DNA, PNA & XNA  2012;3(1):22-30.
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.
doi:10.4161/adna.19906
PMCID: PMC3368813  PMID: 22679530
antisense; cellular delivery; lipoplex; octaarginine (CPP); peptide nucleic acid (PNA); polyethyleneimine (PEI)
31.  A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery 
Artificial DNA, PNA & XNA  2012;3(1):3-4.
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.
doi:10.4161/adna.19843
PMCID: PMC3368814  PMID: 22679527
aptamer; delivery; DNA origami; nanocapsule; nanoscience
32.  (α,α-dimethyl)glycyl (dmg) PNAs 
Artificial DNA, PNA & XNA  2012;3(1):5-13.
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 homothyminePNA2: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.
doi:10.4161/adna.19185
PMCID: PMC3368815  PMID: 22679528
(α,α-dimethyl)glycyl PNA; gem-dimethylglycyl PNA; peptide nucleic acid; PNA-DNA binding; sterically constrained PNA analog; α-aminoisobutyric acid PNA
33.  RNA-DNA sequence differences spell genetic code ambiguities 
Artificial DNA, PNA & XNA  2011;2(3):69-70.
A recent paper in Science by Li et al. 20111 reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized. 
PMCID: PMC3324336  PMID: 22567189
gene expression; RNA editing; RNA-DNA differences; transcription; transcriptome
34.  Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents 
Artificial DNA, PNA & XNA  2011;2(3):71-78.
Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics.
PMCID: PMC3324337  PMID: 22567190
2’-O-Methyl; anti-miR; delivery; Gymnosis; Locked Nucleic Acids; miR-122; miRNA; Peptide Nucleic Acids; phosphorothioate; transfection
35.  PNA HyBeacons for analysis of human mutations related to statin-induced myopathy 
Artificial DNA, PNA & XNA  2011;2(3):79-89.
Aminoalkyl and alkyne-tagged PNA HyBeacons have been synthesized, labeled with fluorescein via conventional amide bond or triazole formation (click chemistry) and used to detect single nucleotide polymorphisms (SNPs) implicated in statin-induced myopathy. The PNA HyBeacons gave much better mismatch/mutant discrimination than conventional DNA HyBeacons but smaller fluorescence changes on melting.
PMCID: PMC3324338  PMID: 22567191
fluorescence melting; genetic analysis; HyBeacon; PNA; SNPs; statin-induced myopathy
36.  Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers 
Artificial DNA, PNA & XNA  2011;2(3):90-99.
We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA length (to adjust binding affinity). In general, the carrier CPP-PNA constructs including the ones with decanoyl modification provided significant increase of the activity of unmodified antisense PNA as well as of antisense octaarginine-PNA conjugates. Antisense activity, and by inference cellular delivery, of unmodified antisense PNA was enhanced at least 20-fold at 6 μM upon the complexation with an equimolar amount of nonamer carrier decanoyl-CPP-PNA (Deca-cPNA1(9)-(D-Arg)8). The antisense activity of a CPP-PNA ((D-Arg)8-asPNA) (at 2 μM) was improved 6-fold and 8-fold by a heptamer carrier CPP-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP-PNA carriers may be used as effective cellular delivery vectors for different types of antisense oligomers and also allows use of combinations of (at least two) different CPP ligands.
PMCID: PMC3324339  PMID: 22567192
antisense; carrier; cell penetrating peptide (CPP); cellular delivery; peptide nucleic acid (PNA); siRNA
37.  Artificial DNA structures 
doi:10.4161/adna.2.2.17085
PMCID: PMC3166487  PMID: 21912724
38.  A ribozyme transcribed by a ribozyme 
Artificial DNA, PNA & XNA  2011;2(2):40-42.
Prominent current ideas on how life emerged on Earth include an RNA world hypothesis in which RNA performed informational as well as catalytic functions in the absence of both DNA and protein. Demonstration of a self-replicative system based on ribonucleic acid polymers as both information carriers and catalysts would lend support to such a scenario. A pivotal component of this system would be an RNA dependent RNA polymerase ribozyme capable of replicating its own RNA gene. Recent work from the Holliger group at the Laboratory for Molecular Biology in Cambridge has provided synthetic ribozymes1 that just might foreshadow the future engineering of such self-replicative systems.
doi:10.4161/adna.2.2.16852
PMCID: PMC3166488  PMID: 21912725
ribozyme; RNA dependent RNA polymerase; In vitro evolution; RNA engineering; transcription
39.  Noncovalent binding and fluorogenic response of cyanine dyes to DNA homoquadruplex and PNA-DNA heteroquadruplex structures 
Artificial DNA, PNA & XNA  2011;2(2):43-49.
Two symmetrical cyanine dyes based on benzothiazole heterocycles and a trimethine bridge were found to bind to a parallel-stranded DNA guanine quadruplex based on the MYC oncogene promoter sequence with high nanomolar affinity and 1:1 stoichiometry. The dyes exhibited substantial fluorescence enhancements upon binding. In the presence of homologous guanine-rich peptide nucleic acid oligomers, PNA-DNA heteroquadruplexes were formed. The dyes retained their ability to bind to the heteroquadruplexes at low micromolar concentrations and with varying fluorescence enhancements, although indeterminate stoichiometries preclude quantitative comparison of the affinities with the DNA homoquadruplex precursor. The difference in fluorescence enhancement between DNA homoquadruplex and PNA-DNA heteroquadruplex allows the dyes to be used as fluorogenic indicators of hybridization in a facile method for determining PNA-DNA stoichiometry.
doi:10.4161/adna.2.2.16339
PMCID: PMC3166489  PMID: 21912726
PNA-DNA heteroquadruplex; cyanine dyes; hybridization; small molecule-quadruplex recognition; fluorescence enhancement
40.  Pyrrolidinyl peptide nucleic acid with α/β-peptide backbone 
Artificial DNA, PNA & XNA  2011;2(2):50-59.
We describe herein a new conformationally constrained analog of PNA carrying an alternating α/β amino acid backbone consisting of (2′R,4′R)-nucleobase-subtituted proline and (1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). The acpcPNA has been synthesized and evaluated for DNA, RNA and self-pairing properties by thermal denaturation experiments. It can form antiparallel hybrids with complementary DNA with high affinity and sequence specificity. Unlike other PNA systems, the thermal stability of acpcPNA·DNA hybrid is largely independent of G+C contents, and is generally higher than that of acpcPNA·RNA hybrid with the same sequence. Thermodynamic parameters analysis suggest that the A·T base pairs in the acpcPNA·DNA hybrids are enthalpically stabilized over G·C pairs. The acpcPNA also shows a hitherto unreported behavior, namely the inability to form self-pairing hybrids. These unusual properties should make the new acpcPNA a potentially useful candidate for various applications including microarray probes and antigene agents.
doi:10.4161/adna.2.2.16340
PMCID: PMC3166490  PMID: 21912727
peptide nucleic acid; nucleic acid; DNA recognition; RNA recognition; pre-organization; foldamer; α/β-peptide
41.  Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads 
Artificial DNA, PNA & XNA  2011;2(2):60-66.
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.
doi:10.4161/adna.2.2.16562
PMCID: PMC3166491  PMID: 21912728
diagnostics; fluorescence microscopy; fluorescent bead; PNA; ribosomal RNA; Trypanosome
42.  Natural Arsenate DNA? 
Artificial DNA, PNA & XNA  2011;2(1):4-5.
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).
doi:10.4161/adna.2.1.15657
PMCID: PMC3116578  PMID: 21686246
Arsenate; DNA; evolution; origin of life; bacteria
43.  Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA) 
Artificial DNA, PNA & XNA  2011;2(1):23-32.
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.
doi:10.4161/adna.2.1.15553
PMCID: PMC3116579  PMID: 21686249
PNA; triplex; gene correction; repair; DNA modification
44.  Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD) 
Artificial DNA, PNA & XNA  2011;2(1):6-15.
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).
doi:10.4161/adna.2.1.15425
PMCID: PMC3116580  PMID: 21686247
antisense; DMD; exon skipping; in vivo; splicing modulation; therapy
45.  A novel pseudo-complementary PNA G-C base pair 
Artificial DNA, PNA & XNA  2011;2(1):33-37.
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).
doi:10.4161/adna.2.1.15554
PMCID: PMC3116581  PMID: 21686250
DNA recognition; hybridization; nucleobases; synthesis; PNA
46.  Molecular computing by PNA:PNA duplex formation 
Artificial DNA, PNA & XNA  2011;2(1):16-22.
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.
doi:10.4161/adna.2.1.15459
PMCID: PMC3116582  PMID: 21686248
molecular computing; SAT problem; PNA:PNA; microarray; fluorescence
47.  DNA breathes Hoogsteen 
Artificial DNA, PNA & XNA  2011;2(1):1-3.
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.
doi:10.4161/adna.2.1.15509
PMCID: PMC3116583  PMID: 21686245
duplex DNA breathing; hoogsteen base pairs; DNA structure; DNA motility
48.  PNA-based microbial pathogen identification and resistance marker detection: an accurate, isothermal rapid assay based on genome-specific features 
Artificial DNA, PNA & XNA  2010;1(2):1-7.
With the rapidly growing availability of the entire genome sequences of microbial pathogens, there is unmet need for increasingly sensitive systems to monitor the gene-specific markers for diagnosis of bacteremia that enables an earlier detection of causative agent and determination of drug resistance. To address these challenges, a novel FISH-type genomic sequence-based molecular technique is proposed that can identify bacteria and simultaneously detect antibiotic resistance markers for rapid and accurate testing of pathogens. The approach is based on a synergistic combination of advanced Peptide Nucleic Acid (PNA)-based technology and signal-enhancing Rolling Circle Amplification (RCA) reaction to achieve a highly specific and sensitive assay. A specific PNA-DNA construct serves as an exceedingly selective and very effective biomarker, while RCA enhances detection sensitivity and provide with a highly multiplexed assay system. Distinct-color fluorescent decorator probes are used to identify about 20-nucleotide-long signature sequences in bacterial genomic DNA and/or key genetic markers of drug resistance in order to identify and characterize various pathogens. The technique's potential and its utility for clinical diagnostics are illustrated by identification of S. aureus with simultaneous discrimination of methicillin-sensitive (MSSA) versus methicillin-resistant (MRSA) strains. Overall these promising results hint to the adoption of PNA-based rapid sensitive detection for diagnosis of other clinically relevant organisms. Thereby, new assay enables significantly earlier administration of appropriate antimicrobial therapy and may, thus have a positive impact on the outcome of the patient.
PMCID: PMC2953854  PMID: 20953307
49.  A pyrenyl-PNA probe for DNA and RNA recognition 
Artificial DNA, PNA & XNA  2010;1(2):83-89.
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.
doi:10.4161/adna.1.2.13899
PMCID: PMC3116571  PMID: 21686243
peptide nucleic acid; pyrene; DNA; RNA; fluorescence
50.  Evolution of synthetic polymers 
Artificial DNA, PNA & XNA  2010;1(2):61-63.
A strategy for the enrichment of a DNA template that encodes a functionalized PNA oligomer is discussed. The method relies on iterated cycles of chemical translation (of the template into PNA), selection (for function), and amplification (of the survivors). Potential restrictions and future perspectives are considered.
doi:10.4161/adna.1.2.13501
PMCID: PMC3116572  PMID: 21686238
chemical evolution; selection; enrichment; DNA template

Results 26-50 (63)