Targeting-moiety conjugated prodrugs have more cancer-specific targeting and reduced background toxicity over traditional chemotherapy. One promising strategy is to conjugate the targeting moiety with a prodrug activating enzyme, termed suicide enzyme, instead of the prodrug itself. The suicide enzyme will catalytically turnover the prodrug into its toxic form, thus providing greater cancer killing than a direct prodrug conjugate. Here, we employ yeast cytosine deaminase (yCD), which converts the non-toxic prodrug, 5-fluorocytosine, into the toxic chemotherapeutic, 5-fluorouracil. To guarantee low nonspecific activity, we employ a split version of yCD which is inactive until the two yCD fragments are brought into close proximity.
We utilize HaloTag technology for efficient covalent linkage of DNA to split yCD proteins and use cancer-specific DNA aptamer switches to control the reconstitution of split yCD. The binding of the DNA aptamer to its cell surface protein target induces a structural shift. This shift exposes single-stranded sequences for hybridization and allows for the split yCD-DNA to be brought together and regain activity. DNA targeting moieties make this strategy modularly adaptable to various targets without disrupting the split yCD design. The switchable aptamer means the system is turned off unless it is bound to a cancer cell, upon which the suicide enzyme is turned on for cancer killing by amplified prodrug activation.