Using home-grown designed telomeric-DNA sequences, called "telobaits," and highly-advanced DNA sequencing technology, scientists can now precisely determine the length of a single telomere from an individual. This technique could potentially be used to measure the impact of lifestyle or drug interventions to tackle ageing-related diseases. Credit: Dr Javier Koh, Duke-NUS Medical School
Telomeres are repeating DNA sequences found at the ends of chromosomes, which help protect the rest of the chromosome from damage over time. The shortening of telomeres is associated with aging and age-related disease, and measurements of telomere length are often used to assess biological aging; however, current methods are typically low-throughput, time-consuming and can only provide an average telomere length for a pool of DNA fragments rather than the lengths of individual telomeres at the nucleotide level. A team led by researchers from the Duke-NUS Medical School and National Heart Center Singapore (NHCS), along with colleagues in Singapore, China and the U.S., has now developed a new accurate method for measuring biological aging, which uses novel ligands and high-throughput sequencing technology to quickly and precisely measure the lengths of individual telomeres from a large pool of DNA fragments.
To enable their new method, the researchers designed DNA sequences they call “telobaits,” which are complementary to sequences found at the ends of telomeres and include unique barcodes to allow increased throughput via multiplexing. The telobaits attach to and enrich the telomeres, and the full telomere length is preserved when the DNA fragments are digested by restriction enzymes Hinfl and Rsal for downstream high-throughput sequencing. Single-molecule real-time (SMRT) sequencing was used to sequence the telomere-containing DNA fragments, allowing telomere length to be determined at the single nucleotide level.
The method was tested on both human cell lines and patient cells. In cell line experiments, the team found that the mean telomere lengths determined through sequencing were consistent with those obtained using terminal restriction fragment (TRF) analysis, which is the current gold standard for telomere length measurement. The sequencing method was also successful in measuring the extremely short telomere lengths found in T24 cells, achieving results consistent with previous studies of this cell line. In patient peripheral blood leukocytes (PBLs), which are commonly used as a biomarker in research related to aging, the sequencing data showed an expected negative correlation between telomere length and patient age. Additionally, the telomere sequencing revealed the presence and diversity of telomeric variant sequences (TVSs) among patients. The varying lengths and sequences of the TVSs – found between the protective, repeating sequence sections of the telomere – cannot be detected using conventional telomere length measurement techniques and could have important implications for individual aging and disease risk. This research was published in Nature Communications.
“This method for telomere length measurement is an important advance in the field of aging research. From the clinical perspective, we view this as a very promising method for understanding clinical diseases associated with aging such as cardiovascular disease,” said senior co-author Angelah Koh, a senior consultant with the Department of Cardiology at NHCS and associate professor with the SingHealth Duke-NUS Cardiovascular Sciences Academic Clinical Program.
The unique, heterogeneous nature of the TVSs found in patient samples could also have implications for human identification in the forensic field, with the possibility that telomere sequences could serve as unique biological identifiers, noted co-senior author Li Shang, from the Duke-NUS Cancer & Stem Cell Biology Program.