Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells.

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

Macrocyclic Peptide-Conjugated Tip for Fast and Selective Molecular Recognition Imaging by High-Speed Atomic Force Microscopy.

Fast and selective recognition of molecules at the nanometer scale without labeling is a much desired but still challenging goal to achieve. Here, we show the use of high-speed atomic force microscopy (HS-AFM) for real-time and real-space recognition of unlabeled membrane receptors using tips conjugated with small synthetic macrocyclic peptides. The single-molecule recognition method is validated by experiments on the human hepatocyte growth factor receptor (hMET), which selectively binds to the macrocyclic peptide aMD4. By testing and comparing aMD4 synthesized with linkers of different lengths and rigidities, we maximize the interaction between the functionalized tip and hMET added to both a mica surface and supported lipid bilayers. Phase contrast imaging by HS-AFM enables us to discriminate nonlabeled hMET against the murine MET homologue, which does not bind to aMD4. Moreover, using ligands and linkers of small size, we achieve minimal deterioration of the spatial resolution in simultaneous topographic imaging. The versatility of macrocyclic peptides in detecting unlimited types of membrane receptors with high selectivity and the fast imaging by HS-AFM broaden the range of future applications of this method for molecular recognition without labeling.