The overall objective of this project is to develop bacteriochlorophyll- carotenoid based molecular beacons (BChl-MB) as prostate cancer (CaP)-specific photosensitizers for photodynamic therapy (PDT). The basic concept underlying this proposal is depicted in Figure 1. We propose to synthesize a molecular beacon (MB) consisting of any antisense oligodeoxynucleotide (AS-ON) sequence complementary to an mRNA specific for the target cancer cells. The MB contains a single- stranded DNA that can form a stem-loop structure. The loop sequence is an AS-ON. The stem is formed by the annealing of two complementary arm sequences that are on either side of the loop sequence. A dye (D) is attached to the end of one arm and a quencher (Q) is similarly attached to the other end. Proximity of D and Q quenches fluorescence and photoreactivity of the dye by energy transfer. In the presence of the tumor specific mRNA, the MB hybridizes with the mRNA, disrupting the hydrogen bonds of the stem. The quencher is removed from the immediate vicinity of the dyne. Upon irradiating with light, the dye emits fluorescence and generates cytotoxic singlet oxygen. The beacon serves both as a directed photodynamic therapy (PDT) agent and as a tumor specific diagnostic agent. We are proposing to synthesize BCh1-Mbs [in Fig. 1 D= bacteriochlorophyll (BCh1), Q= carotenoid (Car)] containing stem-loop AS-ON sequence targeted at the DD3 mRNA.. We have selected DD3 as the molecular target because it is one of the most CaP- specific genes described to date and is highly over-expressed in CaP. Such MB based PDT agents should be CaP-specific. We will focus on the synthesis of the BChl-MB that can hybridize with DD3 mRNA resulting in the significant increase of the fluorescence and the singlet oxygen production. We will develop cell-specific peptides as delivery reagents fort Bchl-MB and confirm that this agent reaches the target sites. Finally, we will validate the concept of MB based PDT agents in two human prostate cancer cell lines. Finally, we will validate the concept of MB based PDT agents in tow human prostate cancer cell lines, LNCaP (over-expressing DD3) and DU145 (not expressing DD3).
