Sequence Specific Triple Helix Forming Molecules

序列特异性三螺旋形成分子

• 批准号: 7117904
• 负责人: BARRY GOLD
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 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/7117904
• 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）：使用三链体​​形成寡核苷酸（TFO）来对特异性结合DNA进行测序是治疗与异常或外源基因表达相关的疾病的有吸引力的策略。由于三链体结构的形成仅限于延伸的高嘌呤序列，因此使用TFO对特异性地将药剂递送至主沟中的能力进行测序是不可能的。这是由于： (i) TFO 的糖-磷酸骨架在主沟中的不对称位置； (ii) 当 TFO 读取互补链上的信息时极性发生变化。结果是同型嘌呤延伸的任何中断都会导致 TFO 的结合亲和力显着降低。据推测，使用具有非天然 C-糖苷 (oligoTRIP) 的 TFO 可以克服这些限制，该 TFO 将通过 Hoogsteen H-键与 DNA 结合，其糖苷键位于大沟的中心，并垂直于 Watson-Crick 双链体 H-债券。对于每个双链体配对方案（抗GC，2-氨基-喹啉-4-基；抗CG，2-氨基-喹啉-5-基；抗AT，2-氨基-喹唑啉-4-基；抗TA，2-氨基-喹唑啉- 5-yl)有一个独特的TRIP碱基。磷酸糖主链和新型杂环碱基的位置被设计为允许oligoTRIP结合混合的嘌呤/嘧啶序列并“以单一方向读取DNA”。该提案的具体目标是：(1) 合成和表征C-糖苷，称为 TRIP，可以轻松组装成寡聚物 (oligoTRIP)，旨在通过三重序列特异性结合在双链 Watson-Crick DNA 的主沟中进行测序螺旋基序，不需要高嘌呤序列或非生理 pH (2) 使用凝胶位移测定、TM 测量以及化学和酶足迹法评估oligoTRIP 的体外结合亲和力和特异性 (3) 表征分子力。影响 DNA 三链体的稳定性和结构，并量化控制三螺旋形成的热力学，包括序列、阳离子和水合的作用 (4)。在oligoTRIP 的 5' 或 3' 末端添加 Fe-EDTA DNA 切割功能，并确定这些分子是否在大 DNA 靶标中产生序列特异性损伤； (5) 对oligoTRIPS和双链体DNA之间形成的三链体进行高分辨率NMR研究。