Peptide Nucleic Acid Microarrays Research Articles

Synthesis and Characterisation of Photolabile SPhNPPOC-Protected (R)-MiniPEG Containing Chiral γ-Peptide Nucleic Acid Monomers

Peptide nucleic acid (PNA) microarrays are expected to be developed as a new generation of gene detection tools. However, poor water solubility and the limitation of the sequence design of achiral PNA probes mainly hinder their application. Accordingly, (R)-diethylene glycol containing a chiral PNA (miniPEG-γPNA) has been developed to solve these problems. Light-directed synthesis is an effective method to fabricate high-density microarrays. Thiophenyl-2-(2-nitrophenyl)propoxycarbonyl (SPhNPPOC) is a newly synthesised photolabile protective group with high photolytic efficiency. Protecting the PNA monomers with SPhNPPOC may improve the preparation process of PNA microarrays by light-directed synthesis in terms of shortening the deprotection time and restraining side reactions. In this article, SPhNPPOC/carbobenzoxy (Cbz)-protected chiral miniPEG-γPNA monomers are synthesised, and the photo-deprotection rate is approximately twice that of a 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC)-protected monomer. The monomers are expected to be used for the efficient and rapid fabrication of chiral miniPEG-γPNA microarrays through a photolithographic strategy.

Light-Directed Synthesis of High-Density Peptide Nucleic Acid Microarrays.

Peptide nucleic acids (PNAs) are a class of nucleic acid mimics that can bind to the complementary DNA or RNA with high specificity and sensitivity. PNA-based microarrays have distinct characteristics and have improved performance in many aspects compared to DNA microarrays. A new set of PNA monomers has been synthesized and used as the building blocks for the preparation of high density PNA microarrays. These monomers have their backbones protected by the photolabile group 2-(2-nitrophenyl)propyloxy carbonyl (NPPOC), and their exocyclic amino groups protected by amide carbonyl groups. A light-directed synthesis system was designed and applied to the in situ synthesis of a PNA microarray with a density of over 10,000 probes per square centimeter. This PNA microarray was able to detect single and multiple base-mismatches correctly with a high discrimination ratio.

Peptide Nucleic Acids (PNAs) Patterning by an Automated Microarray Synthesis System Through Photolithography

Peptide nucleic acids (PNA) microarray assembled with hundreds of unique PNA oligomers has been regarded as a new and mighty competitor of DNA chip in gene analyzing. However, PNA microarray is still a luxury art due to the difficult and laborious chemical synthesis. Herein, we have developed a fully-automated synthesizer for PNA microarray through photolithography. A preactivation mixer was designed and integrated into the synthesizer in order to get rid of the annoying manual process and increase the coupling efficiency of PNA monomers. The PNA patterning model was carried out to check the performance of the automated synthesizer, revealing that an exposure time of 3 min was sufficient for the complete removal of o-nitroveratryloxycarbonyl (NVOC) groups from the synthetic sites with the help of photosensitizer isopropylthioxanthone and the stepwise yield was measured to be about 98.0%, which is comparable with that from conventional fluorenyl-methyloxycarbonyl (FMOC) chemistry. Those results have definitely demonstrated the possibility and capability of this fully-automated synthesizer to fabricate high-quality PNA microarrays.

A PNA microarray platform for miRNA expression profiling using on-chip labeling technology

MicroRNAs (miRNAs) are short, non-coding RNAs that play a critical role in development, metabolism and other fundamental biological processes, and are also important in diseases such as cancer. To study miRNA expression levels in a systematic and parallel manner, we have developed a highly sensitive, specific and reproducible microarray for miRNA expression profiling using the Peptide Nucleic Acids (PNA) probes which have higher affinity and greater specificity for binding to RNA than do DNA probes. We developed a PNA microarray assay that optimizes PNA probe, hybridization conditions, and on-PNA chip labeling method. In this PANArray™ miRNA method, unlabeled RNA is hybridized to the PNA microarray and labeled by enzymatic ligation of pCp-Cy3 on the chip. This assay showed high reproducibility and low cross-hybridization for miRNAs belonging to the let-7 family and the miR-181 family, which differ by a single nucleotide. The PNA microarray (PANArray™ miRNA) is a rapid throughput technology for analyzing expression profiles with high fidelity, and could prove to be a powerful tool for cancer research, diagnosis and prognosic assessment.

Development of peptide nucleic acid (PNA) microarray for identification of Panax species based on the nuclear ribosomal internal transcribed spacer (ITS) and 5.8S rDNA regions

This study describes the identification of Panax species using a peptide nucleic acid (PNA) microarray. P. ginseng, P. quienquefolius, and P. japonicus were distinguished from each other using 5 PNA probes designed based on three single nucleotide polymorphisms (SNPs) detected in internal transcribed spacer (ITS) and 5.8S rDNA regions. Signal intensity comparison between PNA and DNA microarrays revealed that the PNA microarray provides a significantly more stable and specific fluorescent signal intensity than the DNA microarray. Three Panax species identified by the PNA microarray were denoted as barcode numbers depending on their fluorescent signal patterns of each species using 5 PNA probes (PG-ITS-116, PG-ITS-414-1, PG-ITS-414-2, PG-ITS-425-1, and PG-ITS-425-2). P. ginseng, P. quinquefolius, and P. japonicus were denoted as ‘11010’, ‘00202’ and ‘00000’, respectively. The PNA microarray developed in this study will be useful for legitimizing the distribution of ginseng in domestic and foreign ginseng markets.

Patterning of Peptide Nucleic Acids Using Reactive Microcontact Printing

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

Development towards label- and amplification-free genotyping of genomic DNA

Peptide nucleic acid (PNA) microarrays have been used for the analysis of unlabelled DNA molecules by time-of-flight secondary mass spectrometry (ToF-SIMS). PNAs were synthesised with an automated system in multi-well plates followed by the spotting full-length molecules only onto derivatised silicon surfaces using a common split-pin contact printing instrument. After DNA hybridisation, phosphate specific signals were detected and visualised by ToF-SIMS imaging. Because phosphorus is entirely missing in PNAs but is an integral part of nucleic acids, specific signals were only detected on spots at which DNA bound to complementary PNA probes. Combining PNA microarrays with ToF-SIMS detection enables the sensitive analysis of DNA or RNA targets without the need for introducing labels like fluorescent groups.

Synthesis of photolabile o-nitroveratryloxycarbonyl (NVOC) protected peptide nucleic acid monomers

The chemical synthesis of peptide nucleic acid (PNA) monomers was accomplished using various combinations of the o-nitroveratryloxycarbonyl (NVOC) group ( N-aminoethylglycine backbone) and base labile acyl-type nucleobase protecting groups (anisoyl for adenine and cytosine; isobutyryl for guanine), thus offering a photolithographic solid-phase PNA synthetic strategy compatible with photolithographic oligonucleotide synthesis conditions and allowing the in situ synthesis of PNA microarrays in an essentially neutral medium, by avoiding the use of the commonly used deprotection reagents such as trifluoroacetic acid or piperidine. Convenient methods were also explored to prepare 1-(carboxymethyl)-4- N-(4-methoxybenzoyl)cytosine and 9-(carboxymethyl)-2- N-(isobutyryl)guanine with good yields.

Diagnosis of HNF-1 mutations on a PNA zip-code microarray by single base extension

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1α (HNF-1α) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3′ complementarity to the specific mutation site and 5′ complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1α with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.

Development of PNA microarrays for gene diagnostics with TOF-SIMS

We have used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to examine different immobilization processes of peptide nucleic acid (PNA) and their influence on the hybridization efficiency of unlabeled DNA fragments to complementary PNA. Two different approaches have been used to immobilize PNA onto Au surfaces. One method was to immobilize thiolated PNA in a single step reaction to the Au surface via an AuS bond. The other method was to crosslink the N-terminal end of the PNA to a preformed functionalized self-assembly monolayer (SAM). The influence of length and type of spacer molecules, which increase the distance between the biosensor surface and the PNA sequence, on the hybridization efficiency was also investigated. It was found that a greater spacer length leads to an increased hybridization efficiency. DNA could readily be identified by detecting PO 2 − and PO 3 − or other phosphate-containing molecules from the DNA backbone. Using TOF-SIMS a very good discrimination between complementary and non-complementary hybridized DNA sequences was achieved. It can be concluded that TOF-SIMS is a very useful technique for identifying unlabeled hybridized DNA on PNA biosensor chips. This method has the potential for providing a rapid method for DNA diagnostics and is also very suitable for studying the complexity of the immobilization and hybridization processes. Both the absence of a labeling procedure and the increase in the number of phosphate signals with increasing sequence length makes this technique especially useful for directly analyzing genomic DNA.