Telomeres (repetitive sequences located at the ends of chromosomes) have a number of biological functions, including protecting chromosomes from degrading and preventing individual chromosomes from linking to each other. They are also involved in the ageing process and shorten each time a cell replicates. Scientists believe that at least eight to ten DNA base pairs are lost each time a cell divides, because of the so-called end replication problem.

Techniques to analyse telomeres, and in particular those that measure both the DNA base pair length and their biophysical dimensions, are crucial for studying the molecular mechanisms behind these processes, which are directly related to ageing and related diseases. At the moment, however, there are no methods to measure these quantities in individual cell nuclei.

Dual probe contains three essential components

A team of researchers led by Charlie Jeynes of the University of Exeter has now developed a way to do just this using a dual probe that contains three essential components. “The first is a 10 nm sized gold nanoparticle, the second a peptide nucleic acid (PNA) sequence (which is a sequence of nucleic acids that strongly binds to complementary DNA sequences on human chromosomes), and the third is a fluorescent molecule called Alexa 647,” explains Jeynes, who is at the Centre for Biomedical Modelling and Analysis at Exeter. “We designed the PNA so that it specifically binds to the telomere region of a chromosome. The gold nanoparticle and fluorescent molecule, for their part, allow us to measure the biophysical nanometre dimensions of the telomere.”

The researchers indirectly measured the telomere length in base pairs of DNA by counting the number of gold nanoparticles that had attached to the telomere region on human chromosomes. “There is a simple ratio between the numbers of gold nanoparticles and base pairs of DNA that is intrinsic to the probe design,” says Jeynes. “We measured the gold nanoparticles using a technique called X-ray Fluorescence Microanalysis at the Diamond Light Source, which is one the brightest X-ray sources in the world. We needed this intense brightness since it allows us to detect the gold nanoparticles with extreme sensitivity.”

dSTORM measures telomere diameters

Although the spatial resolution of the X-ray beam is poor, the researchers say they overcame this problem by exploiting the fluorescent part of their probe and a super-resolution microscopy technique called dSTORM. This allowed them to measure the actual diameters of the telomeres, which they found to be between 60 and 300 nm.

“Our dual-purpose probe could be used to help answer important biological questions through correlative imaging approaches,” Jeynes tells nanotechweb.org. “This is important and shows that nanotechnology can reveal biophysical processes that conventional techniques struggle with. The method we developed does not just apply to telomeres but could be used to quantify genes in chromosomes and measure how the biophysical properties of these genes affect function.

Revealing fundamental mechanisms within the human chromosome

“For telomere biology, there is a currently a debate about how DNA compaction within the telemetric region affects the mechanisms of cell ageing. Our technique provides a new tool to elucidate how these mechanisms operate.”

The team, reporting its work in ACS Nano DOI: 10.1021/acsnano.7b07064, is now busy developing an even more sensitive probe containing much smaller gold nanoparticles and visualizing how these attach to telomeres using a combination of X-ray fluorescence, cryo-electron microscopy and super-resolution microscopy. “This will allow for unprecedented biophysical measurements of DNA structure within the human chromosome and reveal fundamental biological mechanisms,” says Jeynes.