Sequence Specific Triple Helix Forming Molecules

描述(申请人提供)：使用三链形成寡核苷酸(TFO)与DNA特异结合的序列是治疗与异常或外源基因表达相关的疾病的一种有吸引力的策略。由于三链结构的形成受限于扩展的高嘌呤序列，因此使用TFOS将特定的递送剂排序到主槽中的能力是不可能的。这是由于：(I)TFO的糖-磷酸主链在主槽中的不对称位置；以及(Ii)TFO读取互补链上的信息时极性的变化。其结果是，同质尿伸展的任何中断都会导致TFO结合亲和力的显著下降。假设这些限制可以使用带有非天然C-糖苷(寡糖)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，并垂直于Watson-Crick双链H键。对于每个双链配对方案(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)，有一个独特的Trip碱基。糖磷酸主链和新型杂环碱基的位置被设计成允许寡聚TRIP与混合的嘌呤/嘧啶序列结合，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，它可以很容易地组装成寡聚体(寡聚TRIP)，其设计是通过三螺旋基序与双链Watson-Crick DNA的主槽特异结合，而不需要高嘌呤序列或非生理性pH。(2)通过凝胶移位分析、TM测量、化学足迹和酶足迹等方法评价寡核苷酸的体外结合亲和力和特异性。(3)表征影响寡核苷酸DNA三链稳定性和结构的分子作用力，并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。(4)在寡核苷酸的5‘或3’末端添加Fe-EDTA DNA切割功能，并确定这些分子是否对大的DNA靶标产生序列特异性损伤；以及(5)对寡核苷酸和双链DNA之间形成的三链进行高分辨率核磁共振研究。

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.