Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation.
The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems.
McGregor
A
A
Department of Biochemistry, The University of Newcastle, Newcastle upon Tyne, UK.
Rao
M V
MV
Duckworth
G
G
Stockley
P G
PG
Connolly
B A
BA
D000345:Affinity Labels; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D036022:Capsid Proteins; D015694:Gene Products, rev / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D010880:Piperidines / Q000737:chemistry*; D011485:Protein Binding / Q000528:radiation effects; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013891:Thiouridine / Q000737:chemistry*; D014466:Ultraviolet Rays; D054321:rev Gene Products, Human Immunodeficiency Virus
D000345:Affinity Labels; D036022:Capsid Proteins; D015694:Gene Products, rev; D015335:Molecular Probes; D009843:Oligoribonucleotides; D010880:Piperidines; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D054321:rev Gene Products, Human Immunodeficiency Virus; D013891:Thiouridine