Abstract
Techniques to analyze human telomeres are imperative in studying the molecular mechanism of aging and related diseases. Two important aspects of telomeres are their length in DNA base pairs (bps) and their biophysical nanometer dimensions. However, there are currently no techniques that can simultaneously measure these quantities in individual cell nuclei. Here, we develop and evaluate a telomere “dual” gold nanoparticle-fluorescent probe simultaneously compatible with both X-ray fluorescence (XRF) and super resolution microscopy. We used silver enhancement to independently visualize the spatial locations of gold nanoparticles inside the nuclei, comparing to a standard QFISH (quantitative fluorescence in situ hybridization) probe, and showed good specificity at ∼90%. For sensitivity, we calculated telomere length based on a DNA/gold binding ratio using XRF and compared to quantitative polymerase chain reaction (qPCR) measurements. The sensitivity was low (∼10%), probably because of steric interference prohibiting the relatively large 10 nm gold nanoparticles access to DNA space. We then measured the biophysical characteristics of individual telomeres using super resolution microscopy. Telomeres that have an average length of ∼10 kbps, have diameters ranging between ∼60–300 nm. Further, we treated cells with a telomere-shortening drug and showed there was a small but significant difference in telomere diameter in drug-treated vs control cells. We discuss our results in relation to the current debate surrounding telomere compaction.
Highlights
Telomeres are repetitive sequences located at the ends of chromosomes
The gold nanoparticles are ideal for detection through the characteristic X-ray fluorescence (XRF) signal for the Au L-shell, while Alexa-647 is an organic dye that is widely used in super resolution microscopy
By quantifying the GNP signal, it is possible to infer how many DNA bases there are in a telomere, if we can obtain an estimate of the ratio of GNPs to DNA bases
Summary
They have a number of functions including protecting the chromosomes from degradation and preventing individual chromosomes from linking to each other They are associated with the aging process and shorten with each cell replication cycle.[1] It is accepted that at least 8−10 base pairs (bps) are lost per cell division due to the “end replication problem”,2,3 while many more bps are thought to be eroded due to reactive oxygen species. There are a number of ways to measure telomere length (in base pairs of DNA), while there are very few methods that can measure biophysical characteristics, like volume Methods such as southern hybridization can measure average telomere length in bulk homogenate samples.[7] Bulk methods mask the heterogeneity that could occur at each telomere within a nucleus.[8,9] For individual telomeres, a method known as quantitative fluorescence in situ hybridization (QFISH) uses a short fluorescence oligonucleotide sequence (made of DNA, or more commonly, PNA) probe for the repeat motif found in telomeres, visualized with standard fluorescence microscopy.[10]
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