A Pterin-FAM-TAMRA Tri-color Fluorescence Biosensor to Detect the Level of KRAS Point Mutation.

Monitoring the changes in the level of KRAS point mutation (the concentration fraction of the KRAS point mutated DNA to the total DNA) in clinical treatment progress can guide and greatly improve the personalized therapy and therapeutic evaluation of patients with cancer. In this work, based on FRET fluorescence quenching and apyrimidinic site-induced guanine/pterin specific binding, we developed a pterin-FAM-TAMRA tri-color fluorescence sensing system to assess the level of KRAS point mutation in one step. The responses from TAMRA displayed good and similar linear correlations in the range from 60 nM to 2 μM for all four types of DNA, resulting in a common linear equation related to the T-DNA concentration (NΔFTAMRA = 2.908cT-DNA + 0.364). Meanwhile, the responses from pterin showed excellent selectivity to W-DNA and an excellent linear correlation to the W-DNA in the concentration range from 60 nM to 1 μM (NΔFpterin = -0.364cgDNA-G + 0.034). This biosensor has an effective concentration range for detecting KRAS point mutations. Especially, because the apyrimidinic site-induced guanine/pterin binding is selective for the detection of wild-type DNA, the sensing system can be applied for clinical mutation level detection of all kinds of KRAS point mutations (G → A, G → C and G → T) in blood samples, which is crucial for the personalized therapy and therapeutic evaluation of patients with most KRAS-related cancer types.