DNA repair activity is strongly linked to the formation of cancer and its progression. As a result, the abberant expression of DNA repair enzymes can be used for cancer diagnosis and prognosis. However, current methods to measure activity are slow, laborious and indirect, which prevents clinicians from using these enzymes as biomarkers despite their high relevance to cancer. To overcome this issue, we developed a simple one-step fluorescence-based assay that directly measures activity of a DNA repair enzyme, ALKBH3.
ALKBH3 (also known as prostate cancer antigen 1) was found to be important for the survival of several types of cancers including prostate, non-small cell lung and pancreatic cancer. Clinical studies revealed that patients with high levels of ALKBH3 had a poorer outcome than patients with lower levels, suggesting the enzyme as a predicter for anticancer drug resistance. Knocking down the enzyme reduces resistance leading to improvements in the effect of anticancer drugs, thereby making ALKBH3 a promising therapeutic target.
Figure: A fluorogenic DNA probe monitors DNA repair activity. A DNA oligomer containing the substrate for the DNA repair enzyme ALKBH3 is modified with fluorescent nucleobases that are initially "dark" due to the neighbouring damaged base (left side). Enzymatic repair of the substrate causes fluorescence lighting-up that directly correlates with the activity of ALKBH3 (right side).
We sought to develop a fluorescent chemosensor that could simply measure ALKBH3 activity to provide fast and accurate assessment of cancers in formats commonly used by clinicians (lysates or live cells). The mechanism of ALKBH3 repair involves oxidative demethylation of N1-methylated adenine in DNA. The enzyme uses Fe(II) and molecular oxygen to oxidize the N-methyl group which spontaneously breaks down releasing formaldehyde and restoring native adenine. With this in mind, we constructed a short DNA oligomer containing the damaged base to be repaired, neighboured by fluorescent nucleobases (two pyenes) whose fluorescence were found to be quenched by the damaged base. Upon ALKBH3 repair, a light-up response in cyan fluorescence is produced, directly reflecting the activity of the enzyme.
Having consructed a “real-time” fluorescent chemosensor, we demonstrated its ability to differentiate levels of ALKBH3 activity in cancer cell lysates and in live-cells. Signals can be generated within hours and only requires addtion of the probe to the samples (no workups). We also showed the sensor can be used to screen for potential ALKBH3 inhibitors in formats used for high throughput screening. Overall, our developed sensor will enable biologists to make better clinical connections with ALKBH3 and cancer and aid in the discovery of novel inhibitors for anticancer therapeutics.
Reference
Fluorescence Monitoring of the Oxidative Repair of DNA Alkylation Damage by ALKBH3, a Prostate Cancer Marker; Andrew A. Beharry, Sandrine Lacoste, Timothy R. O’Connor and Eric T. Kool; J. Am. Chem. Soc., 2016, 138 (11), pp 3647–3650; DOI: 10.1021/jacs.6b00986.