Morningstar M L, Kreutzer D A, Essigmann J M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Chem Res Toxicol. 1997 Dec;10(12):1345-50. doi: 10.1021/tx970052a.
Spontaneous oxidative DNA damage occurs as a consequence of aerobic metabolism, lipid peroxidation, immune responses, ionizing radiation, and some chemical oxidants. These processes yield a vast array of oxidized DNA bases and sugars. The existence of significant steady-state levels of oxidized DNA bases in the genome suggests that these lesions are not completely repaired on a biologically relevant time scale and thus may contribute to mutagenesis. In particular, studies have shown that the steady-state levels of 5-hydroxy-2'-deoxycytidine (dC5-OH) and its deamination product, 5-hydroxy-2'-deoxyuridine (dU5-OH), are similar to those found for 7,8-dihydro-8-oxoguanosine, a known highly mutagenic lesion formed by oxidation of guanosine. Structural and biological properties of dC5-OH and dU5-OH have been constrained by the lack of synthetic methodology for oligonucleotides containing these modified bases. A method is described here for the solid-phase synthesis of oligonucleotides containing dC5-OH and dU5-OH. Preparation of each of the required phosphoramidites involved the selective protection of the base 5-hydroxyl group over the deoxyribose 5'- and 3'-hydroxyl groups. The base composition and the incorporation of the adducts into synthetic heptanucleotides were confirmed after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis. Mass spectrometric analysis of the oligonucleotide products by electrospray MS and GC/MS further confirmed their composition. Most significantly, deamination of the dC5-OH oligomer to a putative dU5-OH product during solid-phase DNA synthesis or oligonucleotide deprotection was not detected by any analytical technique employed.
自发性氧化DNA损伤是有氧代谢、脂质过氧化、免疫反应、电离辐射和一些化学氧化剂作用的结果。这些过程会产生大量氧化的DNA碱基和糖。基因组中存在显著的氧化DNA碱基稳态水平，这表明这些损伤在生物学相关的时间尺度上并未完全修复，因此可能导致诱变。特别是，研究表明，5-羟基-2'-脱氧胞苷（dC5-OH）及其脱氨产物5-羟基-2'-脱氧尿苷（dU5-OH）的稳态水平与7,8-二氢-8-氧代鸟苷相似，7,8-二氢-8-氧代鸟苷是鸟苷氧化形成的一种已知的高诱变性损伤。由于缺乏含有这些修饰碱基的寡核苷酸的合成方法，dC5-OH和dU5-OH的结构和生物学特性受到了限制。本文描述了一种固相合成含有dC5-OH和dU5-OH的寡核苷酸的方法。制备每种所需的亚磷酰胺涉及对碱基5-羟基相对于脱氧核糖5'-和3'-羟基的选择性保护。通过酶切和HPLC分析纯化修饰的寡核苷酸后，确认了碱基组成以及加合物掺入合成七核苷酸的情况。通过电喷雾质谱和气相色谱/质谱对寡核苷酸产物进行质谱分析进一步证实了它们的组成。最重要的是，在固相DNA合成或寡核苷酸脱保护过程中，未通过任何所采用的分析技术检测到dC5-OH寡聚物脱氨为假定的dU5-OH产物。
