Sequence Specific Triple Helix Forming Molecules

序列特异性三螺旋形成分子

• 批准号: 6778114
• 负责人: BARRY GOLD
• 金额: $ 29.64万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6778114
• 关键词: calorimetry; chemical kinetics; drug design /synthesis /production; ethylenediaminetetraacetate; gel mobility shift assay; glycosides; hydrogen bond; iron compounds; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; oligonucleotides; thermodynamics; triple helix

DESCRIPTION (provided by applicant): The use triplex forming oligonucleotides (TFO) to sequence specifically bind to DNA is an attractive strategy in the treatment of diseases associated with aberrant or foreign gene expression. The ability to sequence specifically deliver agents into the major groove using TFOs has not been possible because the formation of triplex structures is restricted to extended homopurine sequences. This is a consequence of the: (i) asymmetric location of the TFO's sugar-phosphate backbone in the major groove; and (ii) change in polarity as the TFO reads information on the complementary strands. The result is that any interruption in a homopurine stretch causes a significant decrease in the TFO's binding affinity. It is hypothesized that these limitations can be overcome using TFO's with unnatural C-glycosides (oligoTRIPs) that will bind via Hoogsteen H-bonding to DNA with their glycosidic bond in the center of the major groove and perpendicular to the Watson-Crick duplex H-bonds. For each duplex pairing scheme (antiGC, 2-amino-quinolin-4-yl; antiCG, 2-amino-quinolin-5-yl; antiAT, 2-amino-quinazolin-4-yl; antiTA, 2-amino-quinazolin-5-yl) there is a unique TRIP base. The location of the sugar phosphate backbone and the novel heterocyclic bases are designed to allow the oligoTRIPs to bind to mixed purine/pyrimidine sequences and "read DNA in a single direction. The Specific Aims of the proposal are to: (1) Synthesize and characterize C-glycosides, referred to as TRIPs, that can be readily assembled into oligomers (oligoTRIPs) that are designed to sequence specifically bind in the major groove of duplex Watson-Crick DNA with via a triple helix motif and without a requirement for homopurine sequences or non-physiological pH. (2) Evaluate the in vitro binding affinities and specificities of the oligoTRIPs using gel shift assays, TM measurements, and chemical and enzymatic footprinting. (3) Characterize the molecular forces that influence the stability and structure of DNA triplexes with oligoTRIPs and quantify the thermodynamics governing triple helical formation, including the role of sequence, cations and hydration. (4) Append an Fe-EDTA DNA cleaving functionality on the 5' or 3'-terminus of the oligoTRIPs and determine if these molecules generate sequence specific damage in large DNA targets; and (5) Perform high resolution NMR studies on triplexes formed between oligoTRIPS and duplex DNA.

描述（由申请人提供）：使用三聚体形成寡核苷酸（TFO）对DNA特异性结合进行测序是治疗与异常或外源基因表达相关疾病的一种有吸引力的策略。由于三联体结构的形成仅限于扩展的同嘌呤序列，因此使用tfo对主要凹槽中的药物进行特异性递送的能力是不可能的。这是由于：(i) TFO的糖-磷酸主链在主槽中的位置不对称；当TFO读取互补链上的信息时，极性会发生变化。结果是，同嘌呤拉伸的任何中断都会导致TFO的结合亲和力显著降低。假设这些限制可以通过带有非天然c -糖苷（oligoTRIPs）的TFO来克服，这些TFO将通过Hoogsteen氢键与DNA结合，其糖苷键位于主槽的中心，垂直于沃森-克里克双氢键。对于每一种双相配对方案（antiGC， 2-氨基喹啉-4-基；antig， 2-氨基喹啉-5-基；antiAT， 2-氨基喹啉-4-基；antiTA， 2-氨基喹啉-5-基），都有一个独特的TRIP碱基。磷酸糖骨架和新型杂环碱基的位置被设计成允许寡核苷酸结合到混合嘌呤/嘧啶序列上，并“以单一方向读取DNA”。该建议的具体目标是：(1)合成和表征c -糖苷，称为TRIPs，可以很容易地组装成寡聚物（oligoTRIPs），设计用于通过三螺旋基元在双沃森-克里克DNA的主要凹槽中特异性结合的序列，而不需要同嘌呤序列或非生理ph值。(2)使用凝胶移位测定、TM测量和化学和酶印迹来评估寡聚物的体外结合亲和力和特异性。(3)表征影响寡核苷酸DNA三联体稳定性和结构的分子力，量化控制三螺旋形成的热力学，包括序列、阳离子和水合作用的作用。(4)在寡核苷酸的5‘或3’端附加Fe-EDTA DNA切割功能，并确定这些分子是否在大的DNA靶标中产生序列特异性损伤；(5)对寡核苷酸和双链DNA之间形成的三联体进行高分辨率核磁共振研究。