1 CNRS UPR 1142, Institute of Human Genetics, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5, France, 2 Centre of Assisted Reproduction and Reproductive Genetics, Institute of Biology and Medical Genetics, Motol Hospital, V uvalu 84, 150 06 Praha 5, Czech Republic and 3 Department of Reproductive Biology B, Arnaud de Villeneuve Hospital, 371 avenue du Doyen Gaston Giraud, F-34295 Montpellier Cedex 5, France
4 To whom correspondence should be addressed. Email: franck.pellestor{at}igh.cnrs.fr
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Abstract |
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Key words: aneuploidy/PNADNA/PNAFISH/PNAPCR
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Introduction |
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Recently, a new approach has been introduced in cytogenetics, based on the use of DNA analogues to recognize and bind to specific nucleic acid sequences in situ. These new types of probes, named peptide nucleic acids (PNA), were developed by Nielsen et al. (1991). Originally conceived and utilized as DNA binding reagents for studying the mechanism of double helix invasion, PNA have quickly evolved from basic research to application in biological assays. The PNA-based protocols benefit from the unique physico-chemical properties of these new synthetic molecules, leading to the development of simple and robust assays. Powerful applications of PNA have thus been developed in microbiology, virology and pharmacology, but have also recently emerged in genetics and cytogenetics. This new family of probes might have significant impact on the exploration of chromosomal and genomic aberrations and lead to marked progress in cytogenetic procedures.
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PNA structure and properties |
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PNA as antigene and antisense agents |
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PNA are able to interact with mRNA independently of the RNA secondary structure. Studies on the mechanisms of anti-sense activity have demonstrated that PNA inhibits expression differently from anti-sense oligonucleotides, acting through RNase-H-mediated degradation of the mRNAoligonucleotide hybrid. Since PNA are not substrates for RNAse, their anti-sense effect acts through steric interference of either RNA processing, transport into cytoplasm or translation, caused by binding to the mRNA (Knudsen and Nielsen, 1996).
The good stability of PNA oligomers, their strong binding efficiency as well as their lack of toxicity at even relatively high concentrations suggested that PNA could constitute highly efficient compounds for effective antisense/antigene application. However, despite the initial rapid success of PNA-based approaches in vitro, progress in the use of PNA as tools for regulating gene expression was hampered by the slow cellular uptake of naked PNA by living cells. Subsequent modifications of PNA have led to significant improvements in the uptake of PNA in eukaryotic cells. The delivery into the cell can be speeded up by coupling PNA to DNA oligomers, to receptor ligands or more efficiently to peptides such as liposomes or cell-penetrating peptides that are rapidly internalized by mammalian cells (Pooga et al., 1998; Cutrona et al., 2000
).
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PNAPCR strategies |
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More recently, novel automated real-time PCR has been developed using PNA. In this method, named Q-PNA PCR, a generic quencher labelled PNA (Q-PNA) is hybridized to the 5' tag sequence of a fluorescent dye-labelled DNA primer in order to quench the fluorescence of the primer. During PCR, the Q-PNA is displaced by incorporation of the primer into amplicons and the fluorescence of the dye label is liberated (Fiandaca et al., 2001).
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Solid-phase hybridization techniques |
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The high-affinity binding of PNA oligomers might lead to faster and easier procedures in most standard hybridization techniques, such as southern and northern blotting (Nielsen and Egholm, 1999). An alternative to southern analysis is the PNA pre-gel hybridization process, which significantly simplifies the procedure of southern hybridization. Labelled PNA are then used as probes, allowing hybridization to a denatured double-stranded DNA sample at low ionic strength prior to loading on the gel. This is different from conventional southern blotting where hybridization occurs after gel electrophoresis and membrane transfer. Here, the mixture is directly subjected to electrophoresis for separation of bound and unbound PNA probes. Because of their neutral charge, excess unbound PNA probes do not migrate in an electrical field. The PNADNA hybrids are then blotted onto a nylon membrane and detected using standard chemiluminescent techniques. The method is sensitive enough to detect a single mismatch in a DNA sample (Perry-O'Keefe et al., 1996
).
Likewise hybridization PNA-based biosensor procedures have been developed in which a single-stranded PNA probe is immobilized onto optical or mass-sensitive transducers to detect the complementary strand or corresponding mismatch in a DNA sample solution. The hybridization events are converted into electric signals by the transducers (Jensen et al., 1997).
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PNA as probes for chromosomal analysis |
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The PNAFISH technique was first used for quantitative telomere analysis. The study of telomere behaviour has become a sensitive subject because of telomere involvement in the processes of cancer evolution and cellular senescence. The FISH technique has been successfully utilized for the in situ detection of telomeric repeat sequences in chromosomes of various species, using synthetic oligonucleotide probes, but the efficiency of these probes has not been sufficient to extend this procedure beyond qualitative analysis of repeat telomeric sequences. To monitor telomere length quantitatively, Lansdorp et al. (1996) utilized fluorescein-labelled PNA probes. By comparing fluorescein-labelled DNA, RNA and PNA probes for the detection of telomeric repeat sequences on human metaphase chromosomes, they first showed that PNA probes yielded superior staining of telomeres. The PNAFISH approach allowed the distinction of fluorescence of individual sister chromatid ends and the accurate estimate of individual and global telomere length in metaphase chromosomes of various cultured human haematopoietic cells. Subsequently, telomere PNA probes were used in several in situ studies of cancer and ageing (Zijlman et al., 1997
; Boei et al., 2000
; Mathioudakis et al., 2000
). Since PRINS provided better efficiency than FISH for the identification of repeat telomeric sequences (Krejci and Koch, 1998
), the performance of PNA method for in situ detection and sizing of telomeric repetitive sequences was compared to the PRINS technique. The two techniques were compared on mouse, hamster and human cell lines and the results were identical in terms of labelling efficiency and sensitivity (Serakinci and Koch, 1999
).
Further developments of PNA technology were focused on the improvement of the specificity of PNA probes and the in situ detection of numerical chromosome abnormalities. Chen et al. (1999) reported that PNA probes could discriminate between two centromeric DNA repeat sequences that differ by only a single base pair. Identical results were obtained with PRINS primers (Pellestor et al., 1995
) and oligonucleotide probes (O'Keefe et al., 1996
), but never with standard DNA probes. The identification of chromosomal variation and the analysis of polymorphisms could greatly benefit from the discrimination power of PNA. The procedure of PNA synthesis allows us to consider the further production of allele-specific probes. This will constitute an evident improvement over the current labelling techniques.
Several chromosome-specific PNA probes were designed and tested. Chen et al. (2000) defined short (1518 mer) and specific PNA probes for alpha-satellite domains of nine chromosomes (chromosomes 1, 2, 7, 9, 11, 17, 18, X and Y) and successfully used them on metaphases and interphase nuclei. To demonstrate the potential utility of PNA probes in clinical application, cultured and uncultured amniocyte preparations were also analysed, giving rates of hybridization efficiency of 9097%. Taneja et al. (2001)
tested other PNA probes for chromosomes 1, X and Y, 1822 mer in size and directly labelled with fluorochromes, on normal human lymphocytes and fibroblasts with abnormal chromosome contents. A fast and simple multicolour PNA protocol was utilized, demonstrating the easy use of PNA probes for in situ labelling assays.
Recently, Pellestor et al. (2003) experimented with PNA technology on human sperm. The adaptation of PNA technology to human sperm constituted an interesting challenge because of the particularities of the sperm nucleus in terms of genomic compaction and accessibility of DNA sequences. To estimate and validate the efficiency of PNA labelling on human sperm, comparative estimates of disomy X, Y and 1 were performed on sperm preparations from healthy subjects using multicolour FISH, PRINS and PNA procedures in parallel. An equivalent quality of in situ nuclear labelling and similar disomy rates were obtained with the three methods. However, the hybridization timing of PNA probes (i.e. 45 min) was considerably shortened in comparison with FISH reaction, which requires an overnight hybridization in order to be efficiently completed on sperm preparations. The fast hybridization kinetics of PNA on sperm was similar to the kinetics of PRINS reaction (2030 min). This similarity might be due to the small size of both PNA probes and PRINS primers. These data highlighted the importance of the probe size for in situ sperm labelling, and consequently the great potential of PNA probes for chromosomal screening on difficult biological material.
Lastly, the PNA strategy has been experimented on isolated human oocytes, polar bodies and blastomeres in order to assess the possibility of using PNA probes for preimplantation genetic diagnosis (PGD; Paulasova et al., 2004). Using directly labelled satellite PNA probes for chromosomes 1, 4, 9, 16, 18, X and Y, we have tested simple and sequential multicolour PNA labelling procedures on 34 in vitro unfertilized oocytes and 23 isolated blastomeres. The combined use of PNA and FISH was also investigated and, in a few cases, FISH labelling was utilized as control in parallel with the PNA reactions. Both rates and types of chromosomal abnormalities scored were in good agreement with results of previous FISH studies. This first use of PNA probes on isolated cells confirms the efficiency of PNA technology for in situ chromosomal analysis and demonstrates the feasibility of using PNA on unique cells. This procedure could become an efficient complement to FISH for PGD because of its simplicity, its fast kinetics of hybridization (4560 min) and the high affinity of PNA probes.
All these studies indicated that PNA probes have multiple advantages for the in situ analysis of nucleic acid sequences. Consequently, the PNA hybridization method might develop quickly within the field of in situ labelling methodology and become a powerful complement to FISH and PRINS for in situ chromosomal investigations
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Future developments |
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Interesting new contributions of PNA could come from the development of applications that cannot be performed using DNA probes. Because of their remarkable sequence discrimination, PNA oligomers have great potential in the growing area of whole genome analysis. It is now evident that future genetic and chromosomal investigations will feature increasingly higher-order multiplexing, as indicated by the rapid development of DNA microarrays. The ability to simultaneously assay many molecular signatures in cells constitutes an important challenge for our understanding of functional cell states, single cell versus tissue-level gene expression, mutation occurrence, and also for the future of diagnostic medicine. Technical platforms for arraying probes, hybridization and data analysis are already in place, and DNA microarrays using the CGH technology are already commercially available for the determination of the copy number of all human chromosomes and individual telomeres. Microarray technology appears to be a very powerful and realistic new diagnostic procedure that may be applied to isolated cells. The preliminary results reported by Bermudez et al. (2004) show movement in this direction. The remarkable hybridization properties of PNA (stability and mismatch discrimination) suggest that PNA oligomers may be efficiently incorporated into microarrays and could improve the timing of the whole procedure, especially when applied on single cells (Weiler et al., 1997
). In association with different fluorochromes, short PNA sequences could constitute a new class of genomic biomarkers for microarray platforms and contribute to the next challenge of extending microarray technology to the single cell level and preimplantation diagnosis.
Another promising feature of PNA might be linked to the development of in vivo fluorescence imaging. The ability to introduce fluorescent probes into living cells will allow deeper study of live gene expression and mRNA transfer (Tyagi and Kramer, 1996). This innovative approach will be more easily applied than non-hybridization-based green fluorescent protein (GFP)-fusion protein systems, and the new multi-photon microscopy will provide efficient tools to visualize multiple gene expression patterns in single living cells (Konig, 2000
). Due to their high in vivo stability and resistance to enzymes and the flexibility of their synthesis procedure, PNA oligomers, conjugated to cell-permeable peptides or liposomes, have a great potential for the future of non-invasive medical imaging.
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Acknowledgements |
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Submitted on April 2, 2004; accepted on May 28, 2004.