γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue

Abstract

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes

“…A 10mer γPNA binds to its complementary DNA and RNA targets with mid-fM K d values [12]. The high affinity of γPNA allows it to invade genomic DNA in order to bind its target site, leading to applications in in vivo gene editing [13,14] and telomere FISH [15,16].…”

“…A 10mer γPNA binds to its complementary DNA and RNA targets with mid-fM Kd values [12]. The high affinity of γPNA allows it to invade genomic DNA in order to bind its target site, leading to applications in in vivo gene editing [13,14] and telomere FISH [15,16]. While high affinity is essential for applications requiring invasion of genomic DNA, use of γPNA in other contexts where the target site is more accessible (e.g., antisense) is susceptible to off-target effects due to the tolerance for single mismatches.…”

DNAzyme-Amplified Label-Free Biosensor for the Simple and Sensitive Detection of Pyrophosphatase

“…The fluorophore is quenched internally at the beginning and is later restored when recognized and bound to its target molecule. The restoration of fluorescence depends on the structural switching of fluorophore–quencher-labelled oligonucleotides, which may interfere with the interactions between DNAs and their target molecules [ 18 , 19 , 20 ]. Each target requires a unique fluorophore-labelled oligonucleotide, which has a complicated labelling and purifying process, so its cost is far higher than that of regular oligonucleotides.…”

Special Issue: Molecular Properties and the Applications of Peptide Nucleic Acids

“…This is the case in the paper by Elksens et al [ 5 ], where PNA was decorated with furan moieties to obtain interstrand cross-linking (ICL); the use of backbone-modified PNAs in this case improved the recognition properties and thus the reaction outcome. A series of papers in this Special Issue also report the use of modified PNAs [ 6 , 7 , 8 ]. Some of these were designed over the years in order to show better solubility and better binding abilities, others were designed for the decoration of PNAs with functional moieties; these can bring new properties and thus new applications.…”

“…Two specific papers demonstrate the usefulness of PNA in the detection of rare mutations (Chiuan-Chian Chiou and co-workers) [ 11 ] and in the assessment of the authenticity of medicinal plants (Byeong Cheol Moon and co-workers) [ 12 ]. The use of PNAprobes has been further extended to the detection of DNA/RNA in cells and tissues; the paper by Bruce Armitage and collaborators describes an interesting application, using PNA miniprobes for telomer length measurement [ 6 ]. This field is ready for a massive technology transfer in the rapidly advancing sector of sensors and diagnostic kits, not only for biomedical applications, but also for other sectors such as the rapid screening of the identity of biological items and materials (e.g., food and raw material control).…”