Incorporation of an artificial protease and nuclease at the HIV-1 Tat binding site of trans-activation responsive RNA.
The synthesis of a C-5 modified uridine phosphoramidite which contains a primary amino group protected with Fmoc is described. During cleavage and deprotection of chemically synthesized RNA, the Fmoc protecting group is removed to yield a free amino group at a predetermined position in the RNA sequence that can be covalently modified with any reporter group, small structural probes, and biological molecules. This modified uridine phosphoramidite was used to incorporate a reactive primary amino group at position 24 in the HIV-1 Tat binding site of a trans-activation responsive (TAR) RNA sequence during chemical syntheses. Modified RNA phosphoramidite was incorporated into RNA oligomers with more than 97% coupling efficiencies. RNA containing modified uridine was cleaved from the support, deprotected, and desalted according to standard procedures. After deprotection and gel purification, nuclease digestion and HPLC analysis were performed to confirm the incorporation of C-5-aminouridine into the RNA sequence. The effect of modified uridine on TAR RNA structure was analyzed by CD spectroscopy and protein binding assays. Site-specific incorporation of EDTA was accomplished by treating primary amine bearing TAR RNA with an isothiocyanato derivative of nitrobenzyl-EDTA.
Shah
K
K
Department of Pharmacology, Robert Wood Johnson Medical School-UMDNJ, New Jersey 08854, USA.
Neenhold
H
H
Wang
Z
Z
Rana
T M
TM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002614:Chelating Agents / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism; D005786:Gene Expression Regulation; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D017879:Isothiocyanates / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000138:chemical synthesis*; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D054322:tat Gene Products, Human Immunodeficiency Virus
D002614:Chelating Agents; D015696:Gene Products, tat; D017879:Isothiocyanates; D009943:Organophosphorus Compounds; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D015534:Trans-Activators; C101322:isothiocyanatobenzyl-EDTA; D054322:tat Gene Products, Human Immunodeficiency Virus; C101323:uridine phosphoramidite; C037152:isothiocyanic acid; D004492:Edetic Acid; D012260:Ribonucleases; D010450:Endopeptidases; D014529:Uridine