Abstract
Vitamin D receptor (VDR) ligands, such as 1α,25-dihydroxyvitamin D3 (1α,25(OH) 2D3) and its analogs regulate the expression of multiple genes that play essential roles in calcium and phosphate homeostasis, cell differentiation, and the immune system via VDR. Many vitamin D analogs have been synthesized and studied for clinical uses in the treatment of various diseases. Previously, we developed the LucC-ligand binding domain (LBD)-LucN biosensor using split-luciferase, which can detect and discriminate between VDR agonists and antagonists in a short time. Our aim is to develop a highly sensitive detection system for 1α,25(OH) 2D3 and its analogs in the serum or organs. However, the sensitivity of the developed LucC-LBD-LucN biosensor was not high enough. Therefore, we further developed a novel inter-molecular biosensor designated as LucN-LXXLL+LBD-LucC that showed a high light intensity in response to pM order of 1α,25(OH) 2D3. Additionally, we evaluated the binding affinity of the vitamin D analogs for the wild-type and the rickets-associated mutant R274L of VDR using LucN-LXXLL+LBD(WT)-LucC and LucN-LXXLL+LBD(R274L)-LucC biosensor, respectively.
