Split DNAzyme probe for detection of low concentration of mutated DNA in the excess amount of normal DNA for early-stage cancer diagnosis.

Mackenzie Fey¹, Brittany Mueller¹, and Dmitry M. Kolpashchikov^1,2,3 

1. Chemistry Department, University of Central Florida, Orlando, Fl 32816, USA 

2. National Center for Forensic Science University of Central Florida, Orlando, Florida 32816, USA 

3. Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida 32816, USA 

Mutation in DNA can cause cancer. Non-invasive early-stage cancer diagnosis might be based on detection of the mutated DNA in blood samples. Our goal is to form a universal system that can recognize small levels of DNA with a point mutation while being immersed in a large amount of unmutated DNA. We are exploring this by creating a system that will provide a fluorescent signal upon the binding of mutated EGFR DNA, containing a point mutation for lung cancer, to our signaling sequence while not binding to the wild type EGFR DNA. The system is based on a binary (split) DNAzyme sensor.¹ Our system consists of an F-substrate strand, containing fluorophore and quencher oligonucleotide labeled ends, and a split DNA complex containing a strand labeled DzU, which binds both the F-substrate strand and the EGFR DNA, and a strand labeled DzS, serving the same purpose. We optimized the sequences of DzU and Dzs strands to achieve the highest possible signal-to-background response in the presence of fully mutant DNA analyte. Furthermore, we optimized the system to detect low concentrations of complementary mutant strand in excess amount of single base mismatched wild type DNA sequences. The proposed technology might be useful for early-stage cancer diagnosis.

This work was supported by the National Science Foundation through the CCF: Software and Hardware Foundations under cooperative agreement SHF-2226021.

References

Gerasimova Y. V., Kolpashchikov D. M. (2013) Folding 16S RNA in a signal-producing structure for detection of bacteria. Angew. Chem., 52, 10586-10588.