Shahid Kazi Abdus, Majumdar Alokes, Alam Rowshon, Liu Su-Ting, Kuan Jean Y, Sui Xuifen, Cuenoud Bernard, Glazer Peter M, Miller Paul S, Seidman Michael M
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Biochemistry. 2006 Feb 14;45(6):1970-8. doi: 10.1021/bi0520986.
Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines.
三链螺旋形成寡核苷酸（TFOs）可能作为基因靶向试剂，用于遗传性疾病的“原位”基因治疗。第三链与双链磷酸之间的负电荷排斥以及嘧啶基序第三链中相邻质子化胞嘧啶之间的不稳定正电荷排斥对三链体形成构成挑战。在此，我们描述了旨在靶向人类β-珠蛋白基因中一个位点的TFOs的合成，该位点是β-珠蛋白病（包括镰状细胞贫血）潜在突变的位点。靶点是一个不间断的聚嘌呤：聚嘧啶序列，在补骨脂素交联位点旁边包含四个相邻的胞嘧啶。尽管引入了其他稳定碱基和糖类似物，但含有四个相邻胞嘧啶或5-甲基胞嘧啶的嘧啶基序TFOs在生理pH下并未形成稳定的三链体。我们合成了一系列含有2'-O-甲基（OMe）和2'-O-氨基乙氧基取代（AE）的补骨脂素-TFOs（pso-TFOs），以及作为中性胞嘧啶替代物的8-氧代腺嘌呤（A8）和2'-O-甲基假异胞苷（P）。热稳定性测量表明，含有A8的TFOs不符合先前工作中确立的标准。然而，含有P的TFOs确实形成了具有合适解链温度（Tm）和结合速率常数（k(ON)）值的三链体。一种含有AE和P残基的pso-TFO具有足够的活性，能够通过直接测量靶点处的交联形成来确定其在活细胞中的靶向作用。我们的结果验证了我们先前研究中描述的修饰形式，并表明P取代是解决靶向含有相邻胞嘧啶的基因组序列问题的有效方法。
