| Autor: |
Jeynes JCG; Centre for Biomedical Modelling and Analysis, University of Exeter , Exeter, Devon, U.K. , EX2 5DW., Geraki K; Diamond Light Source , Didcot, Oxfordshire U.K. , OX11 0DE., Jeynes C; Ion Beam Centre, University of Surrey , Guildford, Surrey, U.K. , GU2 7XH., Zhaohong M; Centre for Ion Beam Applications, National University of Singapore , Singapore , 119077., Bettiol AA; Centre for Ion Beam Applications, National University of Singapore , Singapore , 119077., Latorre E; Medical School, RILD Building, University of Exeter , Barrack Road, Exeter, Devon, EX2 5DW., Harries LW; Medical School, RILD Building, University of Exeter , Barrack Road, Exeter, Devon, EX2 5DW., Soeller C; Living Systems Institute & Biomedical Physics, University of Exeter , Exeter, Devon, U.K. , EX2 5DW. |
| Abstrakt: |
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. |
