DNA-Paint-Based Super-Resolution Imaging Of Telomeres And Telomerase

Telomeres are "cap"-like nuclear protein structures located at the ends of chromosomes that maintain genome stability and protect the ends of chromosomal DNAs.Telomeres gradually shorten with the periodic replication of chromosomes,and abnormal changes in telomere length are associated with many diseases.Telomerase is a telomere-specific reverse transcriptase that can synthesize telomere sequences using its own RNA as a template,and has the function of repairing telomere length.More than 85% of tumor cells exhibit telomerase activity.Therefore,the study of telomeres and telomerase is helpful for the early diagnosis and treatment of cancer.Here,a new method for telomerase activity and telomere detection is established using DNA-PAINT(point accumulation for nanoscale topographic imaging).Specific research work includes:1.A new method for quantitative detection of telomerase activity based on DNA-PAINT and q PAINT technology was proposed.We achieved a spatial resolution of about 10 nm.Our method has the following advantages.First,it avoids complicated polymerase chain reaction and electrophoresis procedures.Second,it enables super-resolution imaging of reaction products with high signal-to-noise ratios and facilitates localization of telomere extension sites at the single particle level,resulting in high sensitivity.Third,the detection scheme of the DNA-PAINT strategy allows direct in situ observation of the telomere elongation process,which has never been achieved before.All these advantages make the DNA-PAINT telomerase detection strategy of great significance for the dynamic study of telomerase-related physiological processes and cancer diagnosis.2.A DNA-PAINT-based fluorescence in situ hybridization(FISH)technique was proposed to achieve super-resolution imaging of telomeres in the nucleus.In the experiment,the telomere complementary sequence labeled with Cy5 dye was used as the telomere imaging probe,and the centromere complementary DNA sequence labeled with Cy3 dye was used as the centromere imaging probe,and the telomere and centromere of cells were detected.DNA-PAINT imaging was performed and a commercial peptide nucleic acid(PNA)probe was used as a control.The results showed that the DNA imaging probe used in this paper can meet the requirements of DNA-PAINT super-resolution imaging,and the obtained super-resolution image has a localization accuracy of 25 nm.3.Preliminary exploration of telomere length measurement based on q PAINT was investigated.After DNA-PAINT imaging of telomeres,precise measurement of telomere length can be achieved by q PAINT.A synthetic telomere sequence was used as a standard reference for length measurement,which was anchored to the glass bottom surface of the chambered coverslip by means of biotin-streptavidin conjugation.DNA-PAINT imaging was performed on telomere sequences to explore the switching characteristics of the fluorescence signals of the imaging probes.The results showed that photo-switching of the imaging probes was not active enough,but still showed the potential for accurate measurements of telomere length using this method.