INCORPORATION OF AN ARTIFICIAL PROTEASE AND NUCLEASE AT THE HIV-1 TAT-BINDING SITE OF TRANSACTIVATION RESPONSIVE RNA

The synthesis of a C-5 modified uridine phosphoramidite which contains a primary amino group protected with Fmoc is described. During cleava ge and deprotection of chemically synthesized RNA, the Fmoc protecting group is removed to yield a free amino group at a predetermined posit ion in the RNA sequence that can be covalently modified with any repor ter group, small structural probes, and biological molecules. This mod ified uridine phosphoramidite was used to incorporate a reactive prima ry amino group at position 24 in the HIV-1 Tat binding site of a trans -activation responsive (TAR) RNA sequence during chemical syntheses. M odified RNA phosphoramidite was incorporated into RNA oligomers with m ore than 97% coupling efficiencies. RNA containing modified uridine wa s cleaved from the support, deprotected, and desalted according to sta ndard procedures. After deprotection and gel purification, nuclease di gestion and HPLC analysis were performed to confirm the incorporation of C-5-aminouridine into the RNA sequence. The effect of modified urid ine on TAR RNA structure was analyzed by CD spectroscopy and protein b inding assays. Site-specific incorporation of EDTA was accomplished by treating primary amine bearing TAR RNA with an isothiocyanato derivat ive of nitrobenzyl-EDTA.