Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.
We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.
Jensen
K B
KB
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309, USA.
Atkinson
B L
BL
Willis
M C
MC
Koch
T H
TH
Gold
L
L
D001483:Base Sequence; D017931:DNA Primers; D015694:Gene Products, rev / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D013698:Templates, Genetic; D014466:Ultraviolet Rays; D054321:rev Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015694:Gene Products, rev; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D054321:rev Gene Products, Human Immunodeficiency Virus; D012313:RNA