Sequence Specific Triple Helix Forming Molecules

描述（由申请人提供）：使用三链体形成寡核苷酸（TFO）与DNA序列特异性结合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限于延伸的同型嘌呤序列，因此使用TFO将试剂特异性地测序递送到大沟中的能力是不可能的。这是由于：（i）TFO的糖-磷酸骨架在大沟中的不对称位置;以及（ii）当TFO读取互补链上的信息时极性发生变化。其结果是，任何中断在一个同型嘌呤的延伸导致显着降低TFO的结合亲和力。假设这些限制可以使用具有非天然C-糖苷（寡TRIPs）的TFO来克服，所述非天然C-糖苷（寡TRIPs）将通过Hoogsteen H-键合与DNA结合，其中它们的糖苷键在大沟的中心并且垂直于沃森-克里克双链体H-键。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-基），存在独特的TRIP碱基。糖磷酸骨架和新杂环碱基的位置被设计成允许寡TRIPs结合到混合的嘌呤/嘧啶序列上，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TRIPs，可以很容易地组装成低聚物（oligoTRIPs），其被设计为通过三螺旋基序在双链体沃森-克里克DNA的大沟中序列特异性结合，而不需要高嘌呤序列或非生理pH。使用凝胶位移测定、TM测量以及化学和酶足迹法评估oligoTRIPs的体外结合亲和力和特异性。(3)表征影响寡TRIPs DNA三链体的稳定性和结构的分子力，并量化三螺旋形成的热力学，包括序列，阳离子和水合作用。(4)在寡TRIPs的5'或3'-末端附加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;和（5）对寡TRIPs和双链体DNA之间形成的三链体进行高分辨率NMR研究。

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.