Affinity Interactions Between Insulin and Quadruplex DNA

描述(申请人提供)：这项拟议的研究开启了对胰岛素表达的核机制的新的研究，涉及胰岛素和胰岛素样生长因子(IGF)与人类胰岛素基因启动子的胰岛素连接多态区域(ILPR)中的基因组G-Quartet DNA的直接关联。这项建议是基于发现了G-四重DNA寡核苷酸与ILPR重复序列在体外亲和捕获胰岛素。有报道表明，胰岛素和IGFS的核积累可能在转录调控中发挥作用，而且越来越多的证据表明，基因组DNA中G-四元组的形成具有生物学意义，这一点得到了支持。这一结果可能导致识别治疗糖尿病及其并发症的新的基因组靶点。 该R21方案的具体目的是确定人胰岛素和IGF在体外是否表现出与ILPR变体相对应的G-四重DNA寡核苷酸的特异性亲和力结合。它为利用DNA阵列和MALDI质谱学检测筛选蛋白质与特定寡核苷酸结构基序之间的相互作用提供了一种新的方法。我们将研究稳定阳离子、锌、pH、卟啉络合、分子内和分子间G-四元组形成、串联重复数和蛋白质浓度的影响。当观察到亲和力结合时，将进行溶液研究，以提供结合亲和力的更定量的测量。这些里程碑包括：(1)胰岛素蛋白和G-四重组ILPR变体之间的亲和力比胰岛素蛋白质和非G-四重组ILPR变体之间以及白蛋白和G-四重组ILPR变体之间的亲和力至少高103倍；(2)胰岛素蛋白和G-四重组ILPR变体之间的亲和力比其他G-四重组寡核苷酸至少高10倍；以及(3)不同G-四重组ILPR变体之间胰岛素蛋白质的结合亲和力显著不同。这一结果将导致设计新的亲和结合配体，用于合作的R01提案，以在细胞和转基因动物中进行体内研究。

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.