Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Selection and Characterization of NF-κB RNA Aptamers

DNA-binding transcription factors are often important in disease and are possible targets for inhibition by binding to RNA aptamers. As a model, we are studying the application of RNA aptamer technology to target members of the NF-κB family of transcription factors. These powerful gene regulatory proteins drive inflammatory responses, cue HIV-1 replication, and drive resistance to apoptosis in cancer cells.

The two predominant human NF-κB protein monomers are p50 and p65. Homo- and heterodimers of these proteins regulate many promoters. As we have previously shown to be the case for the p50 homodimer form of NF-κB, the DNA-binding domain of a transcription factor can provide an electrostatically favorable binding site for RNA. Previously our laboratory used in vitro selection to identify an RNA aptamer that binds with high specificity and nanomolar affinity to the p50 homodimer form of the human NF-κB. This RNA aptamer was further refined for improved function using the in vivo yeast 3-hybrid system.

In the present study, we select and characterize three RNA aptamers that target the NF-κB p65 protein. p65 is highly homologous to p50, but is not tightly bound by anti-p50 RNA aptamers. Three aptamers are described, two of which (R1, R2) were selected from a library of RNAs containing 60 random nucleotides, and one of which (D1) was selected from a degenerate library based on the anti-p50 aptamer sequence. All three RNA aptamers bind p65 selectively with nanomolar affinities. We describe the sequences and specificities of these aptamers for p50 and p65 proteins, and characterize minimum functional domains using boundary assays. We are further characterizing and refining anti-p65 RNA aptamers in vivo using the yeast three-hybrid system.

Susan E. Wurster*
L. James Maher, III

Department of Biochemistry and Molecular Biology
Mayo Clinic College of Medicine
Rochester, MN 55906

Phone: 507-284-9098
Fax: 507-284-2053
Email: wurster.susan@mayo.edu