A conformational switch-based fluorescent biosensor for homogeneous detection of telomerase activity

Area of Science:

• Biochemistry
• Molecular Biology
• Biomedical Engineering

Background:

• Telomerase is a crucial biomarker for cancer diagnosis and therapy due to its universal presence in tumors.
• Existing methods for telomerase detection require complex procedures and lack sensitivity for early diagnosis.
• Developing novel, sensitive, and efficient biosensors for telomerase activity is essential for advancing cancer research.

Purpose of the Study:

• To develop a conformational switch-based fluorescence biosensor for sensitive detection of telomerase activity.
• To utilize a redesigned RNA aptamer (Spinach) for enhanced fluorescence signaling.
• To establish a platform for cancer cell line differentiation and evaluation of telomerase inhibition.

Main Methods:

• A modified Spinach RNA aptamer was engineered to exist in an inactive conformation, preventing fluorophore binding.
• Telomerase activity, by adding telomere repeats, triggers a conformational switch in the aptamer, enabling DFHBI binding and fluorescence.
• A target recycling circuit involving Ribonuclease H (RNase H) was implemented for signal amplification.

Main Results:

• The biosensor demonstrated high performance in detecting telomerase activity across a range of 100 to 5 x 10^4 HeLa cells.
• A low detection limit of 100 cells was achieved, indicating significant sensitivity.
• The assay successfully differentiated cancer cell lines from normal cell lines and evaluated telomere-binding ligand inhibition.

Conclusions:

• The developed conformational switch-based fluorescent biosensor offers a sensitive and efficient method for telomerase activity detection.
• This assay serves as a valuable platform for cancer diagnosis, differentiation of cell lines, and assessment of therapeutic efficacy.
• The findings hold significant implications for advancing telomerase-targeted cancer therapy and diagnostics.