Nucleic Acids Research Advance Access published online on September 9, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp728
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Molecular Biology |
Characterization of RNA aptamers that disrupt the RUNX1–CBFβ/DNA complex
1Section of Experimental Haematology, Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds LS9 7TF, 2Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, 3MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH and 4The Department of Hematology, University of Cambridge, Hills Road, Cambridge, CB2 2XY, UK
*To whom correspondence should be addressed. Tel: +44 113 343 8525; Fax: +44 113 343 8502; Email: c.bonifer{at}leeds.ac.uk Correspondence may also be addressed to Peter G. Stockley. Tel: +44 113 343 3092; Fax: +44 113 343 7897; Email: stockley{at}bmb.leeds.ac.uk
Received July 14, 2009. Revised August 17, 2009. Accepted August 17, 2009.
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 β protein (CBFβ). 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–CBFβ 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 (
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 CBFβ, 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–CBFβ complex and thus will further aid exploring the functions of this key transcription factor.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.