Department of Biochemistry, Faculty of Medicine, Aligarh Muslim University, Aligarh 202002, India.
J Zhejiang Univ Sci B. 2013 Jan;14(1):40-6. doi: 10.1631/jzus.B1200015.
Peroxynitrite (ONOO(-)) is a powerful oxidant and nitrosative agent and has in vivo existence. The half life of ONOO(-) at physiological pH is less than 1 s. It can react with nucleic acids, proteins, lipoproteins, saccharides, cardiolipin, etc., and can modify their native structures. Action of ONOO(-), synthesized in the authors' laboratory by a rapid quenched flow process, on structural changes of commercially available RNA was studied by ultraviolet (UV), fluorescence, and agarose gel electrophoresis. Compared to native RNA, the ONOO(-)-modified RNA showed hyperchromicity at 260 nm. Furthermore, the ethidium bromide (EtBr) assisted emission intensities of ONOO(-)-modified RNA samples were found to be lower than the emission intensity of native RNA-EtBr complex. Agarose gel electrophoresis of ONOO(-)-modified RNA showed a gradual decrease in band intensities compared to native RNA, an observation clearly due to the poor intercalation of EtBr with ONOO(-)-modified RNA. Native and ONOO(-)-modified RNA samples were used as an antigen to detect autoantibodies in sera of patients with clinically defined breast cancer. Both direct binding and inhibition enzyme-linked immunosorbent assay (ELISA) confirmed the prevalence of native and 0.8 mmol/L ONOO(-)-modified RNA specific autoantibodies in breast cancer patients. Moreover, the progressive retardation in the mobility of immune complexes formed with native or 0.8 mmol/L ONOO(-)-modified RNA and affinity purified immunoglobulin G (IgG) from sera of breast cancer patients supports the findings of the direct binding and inhibition ELISAs. The peroxynitrite treatment to RNA at a higher concentration appears to have damaged or destroyed the typical epitopes on RNA and thus there was a sharp decrease in autoantibodies binding to 1.4 mmol/L ONOO(-)-modified RNA. It may be interpreted that cellular nitrosative stress can modify and confer immunogenicity on RNA molecules. Higher concentrations of nitrogen reactive species can be detrimental to RNA. However, the emergence of native as well as 0.8 mmol/L ONOO(-)-modified RNA as a novel antigen/substrate for autoantibodies in breast cancer patients indicates that, in future, these molecules might find a place on the panel of antigens for early diagnosis of breast cancer.
过氧亚硝酸盐 (ONOO(-)) 是一种强大的氧化剂和硝化剂，具有体内存在。在生理 pH 值下，ONOO(-) 的半衰期小于 1 秒。它可以与核酸、蛋白质、脂蛋白、糖、心磷脂等反应，并可以修饰它们的天然结构。作者实验室通过快速淬火流动过程合成的 ONOO(-) 对市售 RNA 结构变化的作用通过紫外 (UV)、荧光和琼脂糖凝胶电泳进行了研究。与天然 RNA 相比，ONOO(-) 修饰的 RNA 在 260nm 处显示出增色效应。此外，发现 ONOO(-) 修饰的 RNA 样品的溴化乙锭 (EtBr) 辅助发射强度低于天然 RNA-EtBr 复合物的发射强度。与天然 RNA 相比，ONOO(-) 修饰的 RNA 电泳条带强度逐渐降低，这显然是由于 EtBr 与 ONOO(-) 修饰的 RNA 插入不良所致。天然和 ONOO(-) 修饰的 RNA 样品被用作抗原，以检测临床定义的乳腺癌患者血清中的自身抗体。直接结合和抑制酶联免疫吸附测定 (ELISA) 均证实了乳腺癌患者中天然和 0.8mmol/L ONOO(-) 修饰 RNA 特异性自身抗体的存在。此外，与来自乳腺癌患者的血清中天然或 0.8mmol/L ONOO(-) 修饰 RNA 形成的免疫复合物的迁移率逐渐减慢，支持直接结合和抑制 ELISA 的发现。RNA 用更高浓度的过氧亚硝酸盐处理似乎破坏或破坏了 RNA 上的典型表位，因此与 1.4mmol/L ONOO(-) 修饰 RNA 结合的自身抗体急剧减少。可以解释为细胞硝化应激可以修饰 RNA 并赋予其免疫原性。较高浓度的氮反应性物质可能对 RNA 有害。然而，天然和 0.8mmol/L ONOO(-) 修饰 RNA 作为乳腺癌患者自身抗体的新型抗原/底物的出现表明，在未来，这些分子可能在乳腺癌的早期诊断抗原组中找到一席之地。
