Effect of G-rich Higher Order Structures on Insulin Linked Polymorphism Region

富含G的高阶结构对胰岛素连锁多态性区域的影响

• 批准号: 7994921
• 负责人: Hanbin Mao
• 金额: $ 0.5万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-16 至 2010-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994921
• 关键词: Accounting; Address; American; Americas; Attention; Biochemical; Biological Assay; Caucasians; Caucasoid Race; Centers for Disease Control and Prevention (U.S.); Characteristics; DNA; DNA Sequence; DNA Structure; DNA biosynthesis; Development; Diabetes Mellitus; Diagnosis; Disease; Distant; Fragile X Syndrome; Future; G-Quartets; Gene Expression; Generic Drugs; Genes; Genetic Polymorphism; Genetic Transcription; Genome; Genomics; Goals; Health; Hereditary Disease; Higher Order Chromatin Structure; Human; Human Genome Project; Huntington Disease; Individual; Inherited; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Knowledge; Laboratories; Lasers; Lead; Length; Life; Ligands; Light; Link; Mechanics; Methods; Microsatellite Repeats; Minisatellite Repeats; Minority; Molecular; Morphologic artifacts; Nature; Nucleic Acid Regulatory Sequences; Personal Satisfaction; Play; Population; Porphyrins; Production; Promoter Regions; Proteins; Regulation; Research; Rest; Role; Sampling; Structure; Symptoms; Techniques; Testing; Transcriptional Regulation; X-Ray Crystallography; base; helicase; improved; in vivo; instrument; novel; public health relevance; research study; simulation; single molecule; therapeutic target

DESCRIPTION (provided by applicant): The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM. The secondary and higher order structures in short DNA repeats regions can lead to slippage, for example, by stalling the replication fork. The slippage is one of the primary reasons that lead to length polymorphism. Simulation results have predicted higher order G quadruplex structures in ILPR region. However, they have not been observed experimentally, although similar structures have been found in other G rich regions. It is known that helicase can unwind simple G-quadruplex structures, but fails to do so when these structures are stabilized by certain ligands. Based on these facts, the project proposes that there exist higher order G quadruplex structures that are recalcitrant to the helicase unwinding in the ILPR domain. Since mechanical unfolding of single biomolecules has a unique capability to reveal short-lived intermediate structures, a dual-beam dual-trap laser tweezers instrument is employed to investigate the G quadruplex and its potential higher order structures in the ILPR domain at the single molecular level. For this purpose, DNA constructs containing ILPR fragments will be synthesized using a universally applicable strategy that has been successfully developed in authors' laboratories. To identify the existence of higher order G quadruplex structures, conventional biochemical approaches will be employed. To help future development of therapeutics that targets the ILPR repeats, the interaction between G quadruplexes and various ligands, such as porphyrin and insulin, will be characterized using both single molecule and biochemical approaches. To test whether the RecQ DNA helicase is incapable of unwinding individual higher order G quadruplexes, single molecular assays will be performed using laser tweezers. Although RecQ DNA helicase and ILPR DNA fragments are used in the proposed experiments, the finding from this project will be generic enough to shed light on the cause of length polymorphism that is implicated in various diseases including IDDM, fragile X syndrome and Huntington's disease. PUBLIC HEALTH RELEVANCE The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM.

描述（由申请人提供）：该项目的长期目标旨在改善具有遗传成分的疾病的美国人的健康和福祉，例如胰岛素依赖性糖尿病（IDDM，I型糖尿病），脆弱的X综合征和Huntington的疾病，通过研究短DNA重复的调节机制来研究DNA的重复措施。特别是，该项目探讨了新的研究方法，以研究G四链体的影响，次级DNA结构及其潜在的高阶结构对IDDM中与胰岛素连接的多态性区域（ILPR）的长度多态性的潜在高阶结构。短DNA重复区域中的二级和高阶结构可以通过停滞复制叉来导致滑倒。滑移是导致长度多态性的主要原因之一。模拟结果预测了ILPR区域的高阶G四链体结构。然而，尽管在其他富富富富富富富富层中发现了相似的结构，但尚未通过实验观察到它们。众所周知，解旋酶可以放松简单的G四链体结构，但是当这些结构被某些配体稳定时，就无法做到这一点。基于这些事实，该项目提出，存在较高的G四链体结构，这些结构是在ILPR域中放松的解旋酶的固力酶。由于单个生物分子的机械展开具有揭示短寿命的中间结构的独特能力，因此采用双光束双陷阱激光镊子仪器来研究单个分子水平的ILPR域中的G四肌及其潜在的高阶结构。为此，将使用普遍适用的策略合成包含ILPR片段的DNA构建体，该策略已在作者的实验室中成功开发。为了确定高阶G四链体结构的存在，将采用常规的生化方法。为了帮助未来针对ILPR重复的治疗剂的开发，将使用单分子和生化方法来表征G四链体和各种配体之间的相互作用，例如卟啉和胰岛素。为了测试RECQ DNA解旋酶是否无法放松单个高阶G四链体，将使用激光镊子进行单分子测定。尽管在拟议的实验中使用了RECQ DNA解旋酶和ILPR DNA片段，但该项目的发现将足够通用，以阐明长度多态性的原因，这与包括IDDM，脆弱X综合征和亨廷顿氏病在内的各种疾病有关。该项目的长期目标旨在改善具有遗传性成分的疾病的美国人的健康和福祉，例如胰岛素依赖性糖尿病（IDDM，I型糖尿病），脆弱的X综合征和亨廷顿氏病，通过研究短DNA重复的调节性机制，涉及DNA重复这些疾病的调节机制。特别是，该项目探讨了新的研究方法，以研究G四链体的影响，次级DNA结构及其潜在的高阶结构对IDDM中与胰岛素连接的多态性区域（ILPR）的长度多态性的潜在高阶结构。

项目成果

Non-B DNA structures show diverse conformations and complex transition kinetics comparable to RNA or proteins--a perspective from mechanical unfolding and refolding experiments.

• DOI: 10.1002/tcr.201200021
• 发表时间: 2013-02
• 期刊: Chemical record
• 影响因子: 6.6
• 作者: Zhongbo Yu;H. Mao

Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single-molecule level.

• DOI: 10.1021/ja100944j
• 发表时间: 2010-07-07
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Dhakal, Soma;Schonhoft, Joseph D.;Koirala, Deepak;Yu, Zhongbo;Basu, Soumitra;Mao, Hanbin
• 通讯作者: Mao, Hanbin

A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands.

• DOI: 10.1038/nchem.1126
• 发表时间: 2011-08-28
• 期刊: Nature chemistry
• 影响因子: 21.8

Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events.

• DOI: 10.1021/ja210399h
• 发表时间: 2012-03-21
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Yu, Zhongbo;Gaerig, Vanessa;Cui, Yunxi;Kang, HyunJin;Gokhale, Vijay;Zhao, Yuan;Hurley, Laurence H.;Mao, Hanbin
• 通讯作者: Mao, Hanbin