The proposed research initiates a new line of investigation into nuclear mechanisms of insulin expression that involve direct association of insulin and insulin-like growth factors (IGFs) with genomic G-quartet DNA in the insulin-linked polymorphic region (ILPR) of the human insulin gene promoter. The proposal is based on the discovery of in vitro affinity capture of insulin by G-quartet DNA oligonucleotides with the ILPR repeat sequence. It is supported by reports suggesting that nuclear accumulation of insulin and IGFs may play a role in regulation of transcription, as well as by growing evidence of the biological significance of G-quartet formation in genomic DNA. The results could lead to the identification of new genomic targets for treatment of diabetes and its complications.
The specific aim of this R21 proposal is to determine if human insulin and IGFs exhibit specific, affinity binding in vitro to G-quartet DNA oligonucleotides corresponding to the ILPR variants. It takes a new approach to screening interactions between proteins and particular oligonucleotide structural motifs using DNA arrays and MALDI mass spectrometric detection. Effects of stabilizing cations, zinc, pH, porphyrin complexation, intra- vs. intermolecular G-quartet formation, tandem repeat number and protein concentration will be studied. When affinity binding is observed, solution studies will be preformed to provide a more quantitative measure of binding affinity. Milestones include demonstration of (1) at least 103- fold greater affinity between insulin proteins and G-quartet ILPR variants than between insulin proteins and non-G-quartet ILPR variants, and between albumin and G-quartet ILPR variants, (2) at least 10-fold greater affinity between insulin proteins and G-quartet ILPR variants relative to other G-quartet oligonucleotides, and (3) significant differences in binding affinities of insulin proteins among the different G-quartet ILPR variants. The results will lead to the design of new affinity binding ligands for a collaborative R01 proposal to perform in vivo studies in cells and transgenic animals.
