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Affinity Interactions Between Insulin and Quadruplex DNA

胰岛素和四链体 DNA 之间的亲和力相互作用

基本信息

批准号：
7025052
负责人：
Linda B. Mc GOWN
金额：
$ 21.73万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-03-01 至 2008-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7025052
关键词：
affinity labeling biotechnology genetic promoter element insulin insulinlike growth factor matrix assisted laser desorption ionization microarray technology nucleic acid repetitive sequence oligonucleotides protein binding technology /technique development

项目摘要

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.

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）与人胰岛素基因启动子的胰岛素连锁多态性区域（ILPR）中的基因组G-四联体DNA的直接关联。该建议是基于发现在体外亲和捕获胰岛素的G-四联体DNA寡核苷酸与ILPR重复序列。它得到了报告的支持，表明胰岛素和IGFs的核积累可能在转录调控中发挥作用，以及越来越多的证据表明基因组DNA中G-四联体形成的生物学意义。这些结果可能会导致识别新的基因组靶点，用于治疗糖尿病及其并发症。该R21提案的具体目的是确定人胰岛素和IGF是否在体外表现出与对应于ILPR变体的G-四联体DNA寡核苷酸的特异性亲和力结合。它采用了一种新的方法来筛选蛋白质和特定的寡核苷酸结构基序之间的相互作用，使用DNA阵列和MALDI质谱检测。将研究稳定阳离子、锌、pH、卟啉络合、分子内与分子间G-四重峰形成、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测量。重要性包括证明（1）胰岛素蛋白与G-四重体ILPR变体之间的亲和力比胰岛素蛋白与非G-四重体ILPR变体之间以及白蛋白与G-四重体ILPR变体之间的亲和力大至少103倍，（2）胰岛素蛋白与G-四重体ILPR变体之间的亲和力相对于其它G-四重体寡核苷酸大至少10倍，和（3）在不同的G-四联体ILPR变体之间胰岛素蛋白的结合亲和力的显著差异。这些结果将导致新的亲和结合配体的设计合作R 01的建议，在细胞和转基因动物中进行体内研究。