Affinity Interactions Between Insulin and Quadruplex DNA

描述（由申请人提供）：拟议的研究启动了胰岛素表达核机制的新研究路线，涉及胰岛素和胰岛素样生长因子（IGFs）与人类胰岛素基因启动子胰岛素连锁多态性区域（ILPR）基因组g -四方DNA的直接关联。该建议是基于发现胰岛素的g -四重奏DNA寡核苷酸与ILPR重复序列的体外亲和力捕获。有报道表明，胰岛素和IGFs的核积累可能在转录调节中发挥作用，并且越来越多的证据表明基因组DNA中g -四重奏形成的生物学意义支持这一观点。这一结果可能会导致发现治疗糖尿病及其并发症的新的基因组靶点。

DESCRIPTION (provided by applicant): 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.