Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis

Abstract

GammaPNA oligomers having one or two repeats of the sequence AATCCC were designed to hybridize to DNA having one or more repeats of the complementary TTAGGG sequence found in the human telomere. UV melting curves and surface plasmon resonance experiments demonstrate high affinity and cooperativity for hybridization of these miniprobes to DNA having multiple complementary repeats. Fluorescence spectroscopy for Cy3-labeled miniprobes demonstrate increases in fluorescence intensity for assembling multiple short probes on a DNA target compared with fewer longer probes. The fluorescent γPNA miniprobes were then used to stain telomeres in metaphase chromosomes derived from U2OS cells possessing heterogeneous long telomeres and Jurkat cells harboring homogenous short telomeres. The miniprobes yielded comparable fluorescence intensity to a commercially available PNA 18mer probe in U2OS cells, but significantly brighter fluorescence was observed for telomeres in Jurkat cells. These results suggest that γPNA miniprobes can be effective telomere-staining reagents with applications toward analysis of critically short telomeres, which have been implicated in a range of human diseases.

Oxidative guanine base damage regulates human telomerase activity

“…After three PBS washes, cells were fixed in methanol/acetone (v/v) at −20°C for 10 min or in 2% formaldehyde for 10 min at room temperature for TRF2 immuno-staining, and dehydrated in 70%, 90% and 100% ethanol for 5 min. 100 mg/ml telomeric PNA probe (H 2 N-Lys-(AATCCC) 3 -FITC, PNA Innovations 61 ) was diluted 1:500 in hybridization buffer (70% formamide, 10mM Tris HCl pH 7.5, 1x Maleic Acid buffer, 1x MgCl 2 buffer) and boiled for 5 min at 95°C. Samples were incubated for 10 min on a hot plate at 75°C and then at room temperature for 2 h in the dark.…”

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.…”

Novel bead-based platform for direct detection of unlabelled nucleic acids through Single Nucleobase Labelling

“…To achieve this, PNA building blocks containing propanoic acid chains at gamma positions with S-configuration were chosen (Fig. 3) [46][47][48].…”