MODIFIED NUCLEOSIDES IN TRIPLEX FORMING OLIGONUCLEOTIDES

描述(改编自申请人的摘要)：本报告的主要目标 研究是从化学上提炼一类序列特异的DNA结合， 三螺旋形成寡核苷酸(TFOS)。这些产品的独特之处在于 TFOS是第三链与其双链DNA靶标结合pH为 独立，并由ggc和tat三元组稳定，因此 择优取向使结合的TFO相对于 底层双链体中的嘌呤含量越高。与……一致 分子模拟，将2‘-脱氧甲酰霉素(DF)引入3’-胺 在参与氢键的位置修改TFOS，其中 多嘌呤/多嘧啶在双链靶中的运行被CG打断 倒置。这种DF-TFO的结合亲和力是通过条带来评估的 移位分析和定量DNase I足迹技术。这个 用局部高亲和力评价DF对三链结构的影响 铜二氮杂菲裂解。 在初步研究中，3‘A-PRE2ap的同源物显示出10倍于 结合亲和力增加，因为在三个位点包含了DF 与其未经修改的对应产品相比。在第一阶段的研究中，我们会 确认这些发现，并扩大在TFOS中使用DF替代 它们是针对表皮生长因子受体(EGFR)(EG36ap)的， 小鼠胰岛素受体(IR)启动子(IR2ap)和HIV-1(HIV38p)结构域。 如果DF取代改善了三螺旋结构和结合亲和力， 在第二阶段研究中，将评估这种TFOS的生物学效应。

DESCRIPTION (Adapted from applicant's abstract):The primary goal of this study is to chemically refine class of sequence-specific, DNA binding, triple helix-forming oligonucleotides (TFOs). The unique feature of these TFOs is that binding of the third strand to its duplex DNA target is pH independent and is stabilized by GGC and TAT triplets such that the preferred orientation places the bound TFO antiparallel with respect to the more purine rich strand of the underlying duplex. In accord with molecular modeling, 2'-deoxyformycin (df) is introduced into 3'-amine modified TFOs at sites engaged in H-bonding where polypurine/polypyrimidine runs in a duplex target are interrupted by a CG inversion. The binding affinity of such a dF-TFO is assessed by band shift analysis and by quantitative DNase I footprinting techniques. The effect of dF upon triplex structure is assessed by local high affinity Cuphenanthroline cleavage. In preliminary studies, a homologue of 3'A-PRE2ap displayed a 10-fold increase in binding affinity due to inclusion of dF at three sites as compared to its unmodified counterpart. In Phase I studies we will confirm these findings and extend the use of dF substitution within TFOs which are specific for epidermal growth factor receptor (EGFR) (EG36ap), mouse insulin receptor (IR) promoter (IR2ap) and HIV-1 (HIV38p) domains. If dF substitution improves triple helix structure and binding affinity, in Phase II Studies the biological effects of such TFOs will be assessed.