Over 40% of people are likely to be diagnosed with cancer in their lifetimes. Early detection of cancer is instrumental for higher survivability, quality of life, and remission. Certain circulating nucleic acids have been identified as biomarkers for cancer. While amplification techniques like RT-PCR are available, false positives are common limitations.
Developing sensors to confidently detect nucleic acid biomarkers in blood at low concentrations is key in advancing early detection capabilities and treatment options.

Fluorescence resonance energy transfer, or FRET, is a distance dependent, non-radiative, energy transfer process that occurs at the nanometer length scale, well suited for studying the dynamics of DNA molecules. In this project, to detect such biomarkers in solution, fluorophore-labeled DNA constructs based on the Holliday Junction were designed and tested. The utility of Holliday Junction-based DNA sensors arises when combined with the distance dependent nature of FRET. When the four-way junction is complete (i.e. when the target is bound), the changing conformation of the sensor alters the inter-fluorophore distance, leading to a dynamic E_FRET pattern that can be observed in the TIRF microscopy.

Following immobilization of these sensors on the microscope slide and addition of the biomarker target, the sensors were visualized via TIRF microscopy, and their behavior were analyzed using MATLAB. DNA constructs with bound targets were found to display dynamic behavior, identifiable by characteristic fluorescence and FRET traces. Through this method, a clear and precise detection of circulating tumor nucleic acids is possible, opening up a new frontier to cancer screening.