Telo Miniprobes®

Telo Miniprobes®: Illuminating Telomeric DNA

There is growing awareness of the relationship between telomere length and human health. Telomere lengths are commonly determined using PNA fluorescent in situ hybridization (FISH) probes [1]. The repetitive nature of the telomere sequence allows multiple copies of a single probe to hybridize to a given telomere. Thus, the brightness of the fluorescence is determined by the number of hybridized probes, which is determined, in turn, by the length of the telomere. Improving probe brightness is particularly important for detection of critically short telomeres.

Brighter Imaging Under Gentler Conditions

PNA Innovations’ Telo Miniprobes® enable brighter imaging and detection of telomeric DNA [2]. The high affinity of gammaPNA allows shorter probes to be used, leading to hybridization of a greater number of dye-labeled probes to a given target (Figure 1).

 

Telominiprobe

Figure 1. (Left) The Telo Miniprobe® concept. The shorter length of gammaPNA miniprobes allows more probes to bind to a given telomere, leading to brighter fluorescence and fewer signal free ends. (Right) Metaphase chromosome spreads showing staining by a Cy3-labeled Telo Miniprobe® (yellow spots), with DAPI counterstaining (blue).

The high affinity of our Telo Miniprobes® also allows hybridization under gentler nondenaturing conditions [2], which should facilitate concurrent telomere FISH and immunofluorescent staining.

 

Elimination of Washing Steps Using FRET Telo Miniprobes®

The shorter lengths of PNA Innovations’ Telo Miniprobes® also enables the use of miniprobe pairs for FRET imaging of telomeric DNA (Figure 2). Staining with a Telo Miniprobe® pair suitably labeled with donor (e.g. Cy3) and acceptor (e.g. Cy5) dyes leads to efficient FRET only when the two probes are hybridized in close proximity on a telomeric DNA template. Hybridization of longer probes would increase the distance between the donor and acceptor dyes, diminishing the FRET efficiency and, therefore, signal intensity in the FRET channel (e.g. Cy3 excitation/Cy5 emission). Furthermore, while the individual miniprobes are fluorescent, background signal from unhybridized miniprobes is greatly reduced in the FRET channel due to the large distance between the donor and acceptor miniprobes, eliminating the need for washing steps to remove unhybridized probe.

Miniprobe FRET

Figure 2. (Left) Telomeric DNA staining using Telo Miniprobe® FRET pairs. Co-hybridization of donor- and acceptor-labeled gammaPNA Telo Miniprobes® allows detection of telomeric DNA without washing to remove unhybridized miniprobes. (Right) Images of an interphase nucleus from BJ skin fibroblast cells stained with Telo Miniprobe® FRET pairs obtained without washing to remove unhybridized probe. The individual probe channels (Cy3/Cy3 and Cy5/Cy5) show significant background due to unhybridized probe, whereas background is substantially reduced in the FRET channel (Cy3/Cy5), allowing clear imaging of telomere foci. Cells were counterstained with DAPI.

 

References

1. Lansdorp, P. M., Verwoerd, N. P., van de Rijke, F. M., Dragowska, V., Little, M.-T., Dirks, R. W., Raap, A. K. and Tanke, H. J. (1996) Heterogeneity in telomere length of human chromosomes. Hum. Mol. Gen. 5:685-691. PMID: 8733138

2. Pham, H. H., Murphy, C. T., Sureshkumar, G., Ly, D. H., Opresko, P. L. and Armitage, B. A. (2014) Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis Org. Biomol. Chem. 12:7345-7354. PMID: 25115693

 

 

Navigation