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1.  Introducing Artificial DNA: PNA & XNA 
doi:10.4161/adna.1.1.12932
PMCID: PMC3109443  PMID: 21687520
2.  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
3.  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
4.  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
5.  PNA-based microbial pathogen identification and resistance marker detection 
Artificial DNA, PNA & XNA  2010;1(2):76-82.
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.
doi:10.4161/adna.1.2.13256
PMCID: PMC3116573  PMID: 21686242
PNA; bacteral detection; drug resistance; S. aureus; RCA
6.  Hydrogelation abilities of nucleobase-modified cytidines possessing substituted triazoles 
Artificial DNA, PNA & XNA  2010;1(2):90-95.
Nucleoside-derived hydrogelators have been sought for their potential biomedical applications, such as are found in tissue engineering and drug delivery. By judiciously adding a degree of hydrophobicity certain analogues are able to form micelles, bi-layers and gels in water. Research in this area has yet to lay down solid ground rules for the rational design of novel nucleoside gelators making further studies necessary. The synthesis and examination of a series of aryl-substituted 5-triazolylcytidines yielded an analogue that gelates water. 5-(1-(2,2′-bithiophen-3-yl)-1H-1,2,3-triazol-4-yl)-2′-deoxycytidine was found to form gels in water down to 0.3 wt%. The ribocytidine analogue failed to form gel in aqueous solution; but was able to form a hydrogel in the presence of guanosine. Images obtained by SEM show the different architectures of the gel; varying from cribriform to fibrous to lamellar. The present gelating compound studied may have potential as a component of a controlled-release drug delivery system.
doi:10.4161/adna.1.2.13975
PMCID: PMC3116574  PMID: 21686244
hydrogel; nucleoside analogue; SEM; click chemistry; fluorescence
7.  1,4-linked 1,2,3-Triazole des-peptidic analogues of PNA (TzNA) 
Artificial DNA, PNA & XNA  2010;1(2):68-75.
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 PNA2:DNA triplexes. With increasing number of triazole units in the backbone, single stranded PNA oligomers exhibit enhanced self-ordering.
doi:10.4161/adna.1.2.13185
PMCID: PMC3116575  PMID: 21686241
PNA analogues; solid phase click chemistry; triazolyl oligimers; XNA; chimeric PNA-XNA; triazole PNA
8.  Adding mRNA to the list of spatially organized components in bacteria 
Artificial DNA, PNA & XNA  2010;1(2):66-67.
Using LNA in situ hybridization, select mRNAs have been shown to be spatially confined to their chromosomal loci in two distantly related bacterial organisms. Translating ribosomes are diffusion limited by mRNA association.
doi:10.4161/adna.1.2.14150
PMCID: PMC3116576  PMID: 21686240
mRNA spatial distribution; locked nucleic acid (LNA); fluorescence in situ hybridization (FISH)
9.  Small RNAs hit a new target 
Artificial DNA, PNA & XNA  2010;1(2):64-65.
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.
doi:10.4161/adna.1.2.13945
PMCID: PMC3116577  PMID: 21686239
small RNAs; 3′ non-coding transcripts; gene expression modulation; DNA looping; peptide nucleic acid (PNA)
10.  Improving gene silencing of siRNAs via tricyclo-DNA modification 
Artificial DNA, PNA & XNA  2010;1(1):9-16.
Small interfering RNAs (siRNAs) can be exploited for the selective silencing of disease-related genes via the RNA interference (RNAi) machinery and therefore raise hope for future therapeutic applications. Especially chemically modified siRNAs are of interest as they are expected to convert lead siRNA sequences into effective drugs. To study the potential of tricyclo-DNA (tc-DNA) in this context we systematically incorporated tc-DNA units at various positions in a siRNA duplex targeted to the EGFP gene that was expressed in HeLa cells. Silencing activity was measured by FACS, mRNA levels were determined by RT-PCR and the biostability of the modifed siRNAs was determined in human serum. We found that modifications in the 3′-overhangs in both the sense and antisense strands were compatible with the RNAi machinery leading to similar activities compared to wild-type (wt) siRNA. Additional modifications at the 3′-end, the 5′-end and in the center of the sense (passenger) strand were also well tolerated and did not compromise activity. Extensive modifications of the 3′- and the 5′-end in the antisense (guide) strand, however, abolished RNAi activity. Interestingly, modifications in the center of the duplex on both strands, corresponding to the position of the cleavage site by AGO2, increased efficacy relative to wt by a factor of 4 at the lowest concentrations (2 nM) investigated. In all cases, reduction of EGFP fluorescence was accompanied with a reduction of the EGFP mRNA level. Serum stability analysis further showed that 3′-overhang modifications only moderately increased stability while more extensive substitution by tc-DNA residues significantly enhanced biostability.
doi:10.4161/adna.1.1.11385
PMCID: PMC3109438  PMID: 21687522
siRNA; oligonucleotides; tc-DNA; RNA interference; antisense
11.  Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs 
Artificial DNA, PNA & XNA  2010;1(1):17-26.
Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.
doi:10.4161/adna.1.1.11981
PMCID: PMC3109439  PMID: 21687523
altritol; siRNA; RNAi; HBV; lipoplex; non viral vector
12.  Direct observation of two cyclohexenyl (CeNA) ring conformations in duplex DNA 
Artificial DNA, PNA & XNA  2010;1(1):2-8.
Cyclohexene Nucleic Acids (CeNA), in which the 2′-deoxyribofuranose ring of the DNA building blocks is substituted by a cyclohexenyl ring, were designed as potential mimics of natural nucleic acids for antisense and, later, for siRNA applications. CeNA units, in contrast to HNA (hexitol nucleic acid) building blocks, show more flexibility at the level of the C2′–C3′ bond due to the possibility of the cyclohexenyl moiety to adopt different conformations. In order to analyze the influence of CeNA residues onto the helix conformation and hydration of natural nucleic acid structures and to verify the cyclohexenyl ring conformation, a cyclohexenyl-thymine building block was incorporated into the non-self-complementary sequence d(GCG(xT)GCG)/d(CGCACGC) with (xT) a cyclohexene residue. The crystal structure of this sequence has been determined to a resolution of 1.17 Å and contains two duplexes in the asymmetric unit. The global helices belong to the B-type family and the conformations of the cyclohexenyl rings in both duplexes are different. The cyclohexene ring adopts as well the 2H3-conformation (similar to C2′-endo) as the 3H2-conformation (similar to C3′-endo). The crystal packing is stabilized by cobalt hexamine residues and triplet formation.
doi:10.4161/adna.1.1.10952
PMCID: PMC3109440  PMID: 21687521
cyclohexene nucleic acids; CeNA; cobalt hexamine; triplet formation; ring conformation
13.  Natural - synthetic - artificial! 
Artificial DNA, PNA & XNA  2010;1(1):58-59.
The terms “natural,” “synthetic” and “artificial” are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life.
doi:10.4161/adna.1.1.12934
PMCID: PMC3109441  PMID: 21687528
synthetic chromosomes; synthetic cells; artificial cells; artificial life
14.  Mega-cloning and the advent of synthetic genomes 
Artificial DNA, PNA & XNA  2010;1(1):54-57.
Molecular biology owes its prominent role in the biological sciences to the tools of recombinant DNA. While the foundations of recombinant DNA were laid in the 1970s with the discovery of type II restriction endonucleases,1,2 development of robust sequencing technology3 and pioneering work on gene synthesis,4,5 it was not until the turn of the new millennium before the first complete synthetic viral genomes saw the light of day including that of hepatitis C,6 poliovirus,7 and bacteriophage PhiX174.8 Recombinant DNA has come of age as entire cellular genomes are sequenced and stored as digitized information. So what's next? One novel branch of recombinant DNA, referred to as synthetic genomics,9 is occupied with (re)construction of entire cellular genomes from virtual sequence information and using chemical components. Here we look at the most recent developments in such de novo construction. For a broader and more extensive review on genome engineering, the reader is referred to the excellent paper by Carr and Church.10
doi:10.4161/adna.1.1.12935
PMCID: PMC3109442  PMID: 21687527
synthetic genomics; recombinant DNA; genome transplantation; whole-genome assembly; synthetic chromosome
15.  In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts 
Artificial DNA, PNA & XNA  2010;1(1):36-44.
The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and reduce off-target effects enabling possible therapeutic use of siRNA. Recently a large scale direct comparison of the impact of 21 different types of novel chemical modifications on siRNA efficiency and cell viability was published.1 It was found that several types of chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA in vitro. In addition, a novel siRNA design, termed small internally segmented interfering RNA (sisiRNA), composed of an intact antisense strand and segmented guide strand stabilized using LNA was shown to be effective in cell based assays. In the present study we examined the in vivo efficacy of the LNA and UNA modified siRNA and sisiRNA in a mouse model bearing human tumor xenografts. We studied the biodistribution and efficacy of target knockdown in the mouse model. In addition we used whole genome profiling to assess the off-target effects in the liver of the mouse and the tumor xenografts. We report that LNA and UNA modified siRNA and sisiRNA improve the efficacy in target knockdown as compared with unmodified siRNA in the tumor xenografts without formulation. However, the level of off-target gene regulation in both the tumor and the liver correlated with the increase in efficacy in target knockdown, unless the seed region of the siRNA was modified.
doi:10.4161/adna.1.1.12204
PMCID: PMC3109444  PMID: 21687525
LNA; unlocked nucleic acid; siRNA; sisiRNA; off-target effects
16.  Sequence specificity at targeting double-stranded DNA with a γ-PNA oligomer modified with guanidinium G-clamp nucleobases 
Artificial DNA, PNA & XNA  2010;1(1):45-53.
γ-PNA, a new class of peptide nucleic acids, promises to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNA. To check the potential of γ-PNA, we have synthesized a biotinylated, pentadecameric γ-PNA of mixed sequence carrying three guanidinium G-clamp nucleobases. We have found that strand invasion reactions of the γ-PNA oligomer to its fully complementary target within dsDNA occurs with significantly higher binding rates than to targets containing single mismatches. Association of the PNA oligomer to mismatched targets does not go to completion but instead reaches a stationary level at or below 60%, even at conditions of very low ionic strength. Initial binding rates to both matched and mismatched targets experience a steep decrease with increasing salt concentration. We demonstrate that a linear DNA target fragment with the correct target sequence can be purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads. Similarly, supercoiled plasmid DNA is obtained with high purity from an initial sample mixture that included a linear DNA fragment with the fully complementary sequence. Based on the results obtained in this study we believe that γ-PNA has a great potential for specific targeting of chosen duplex DNA sites in a sequence-unrestricted fashion.
doi:10.4161/adna.1.1.12444
PMCID: PMC3109445  PMID: 21687526
strand-invasion; gamma-PNA; duplex DNA recognition; duplex DNA capture; plasmid DNA purification
17.  Peptide nucleic acid probes with charged photocleavable mass markers 
Artificial DNA, PNA & XNA  2010;1(1):27-35.
Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA.
doi:10.4161/adna.1.1.12199
PMCID: PMC3109446  PMID: 21687524
peptide nucleic acid; photocleavable mass marker tag; genetic analysis; halogen-labelled peptide organic acid; SNP

Results 1-17 (17)