Steric block high affinity oligonucleotide analogues: a new tool for mapping RNA-protein binding sites.
Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.
Brown
Douglas
D
Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom.
Joy
Emily
E
Greatorex
Jane
J
Gait
Michael J
MJ
Lever
Andrew M L
AM
D001665:Binding Sites / Q000502:physiology; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D015347:RNA Probes / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology*
D015683:Gene Products, gag; D009843:Oligoribonucleotides; D015347:RNA Probes; D012367:RNA, Viral; D016601:RNA-Binding Proteins