title: Characterization of RNA aptamers that disrupt the RUNX1-CBFbeta/DNA complex.
abstract: The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor beta protein (CBFbeta). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD-CBFbeta complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity ( approximately 100-160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFbeta, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD-CBFbeta complex and thus will further aid exploring the functions of this key transcription factor.
authors.affiliation: Section of Experimental Haematology, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds LS9 7TF, UK.
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Barton
authors.forename: David H J
authors.initials: DH
authors.lastname: Bunka
authors.forename: Stuart E
authors.initials: SE
authors.lastname: Knowling
authors.forename: Pascal
authors.initials: P
authors.lastname: Lefevre
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Warren
authors.forename: Constanze
authors.initials: C
authors.lastname: Bonifer
authors.forename: Peter G
authors.initials: PG
authors.lastname: Stockley
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001665:Binding Sites; D050676:Core Binding Factor Alpha 2 Subunit / Q000037:antagonists & inhibitors*; D050658:Core Binding Factor beta Subunit / Q000378:metabolism*; D004247:DNA / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D050676:Core Binding Factor Alpha 2 Subunit; D050658:Core Binding Factor beta Subunit; D004247:DNA