Hjelstuen O K, Maelandsmo G M, Tønnesen H H, Bremmer P O, Verbruggen A M
Department of Pharmaceutics, University of Oslo, Norway.
Nucl Med Commun. 1998 Aug;19(8):803-12. doi: 10.1097/00006231-199808000-00012.
Oligodeoxynucleotides (ODNs) labelled with an appropriate radionuclide could provide a means to identify serious diseases early on and thereby help initiate treatment at a very early phase. Regardless of important issues like in-vivo stability and membrane passage, the key issue for the oligonucleotide approach is the ability of the radiolabelled ODN to hybridize to the target mRNA. The secondary structure of mRNA does not permit all complementary ODNs to hybridize and a careful selection of the probe with consecutive testing is therefore necessary. This study was initiated to demonstrate hybridization of a 99Tcm-labelled 20-mer ODN to RNA of CAPL (S100A4), a gene reported to be overexpressed in metastatic cancers like breast carcinoma and osteosarcoma. The phosphodiester ODN GX-1 (antisense) and two control sequences (scrambled and random) were conjugated to the bifunctional chelating agent S-benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) and labelled with 99Tcm. The radiolabelled ODNs were purified on a C18 mini-column and characterized on a reverse-phase HPLC system. The radio-chemical purity was > 90% and the product was stable for > 6 h in aqueous medium. The hydrization properties of unlabelled, 32P-labelled and 99Tcm-labelled ODNs to transcribed RNA were studied using polyacrylamide gel electrophoresis (PAGE). Direct hybridization of GX-1 to transcribed RNA was demonstrated. A 50-fold excess of unlabelled ODN over transcribed RNA caused a near to complete consumption of RNA by RNase H activation. In 1:1 proportions of radiolabelled (32P and 99Tcm) ODNs to RNA, only radiolabelled GX-1 was found to hybridize to RNA in a PAGE system. The radiolabelled control ODNs did not show signs of hybridization. This study demonstrates that 3'-99Tcm-labelling of ODNs does not interfere with the hybridization properties of the ODNs in solution, making 99Tcm-labelling an attractive procedure for the future development of antisense technology in imaging.
用适当放射性核素标记的寡脱氧核苷酸（ODN）可为早期识别严重疾病提供一种手段，从而有助于在极早期阶段启动治疗。无论体内稳定性和细胞膜通透性等重要问题如何，寡核苷酸方法的关键问题是放射性标记的ODN与靶mRNA杂交的能力。mRNA的二级结构不允许所有互补的ODN杂交，因此需要仔细选择探针并进行连续测试。启动本研究是为了证明99锝标记的20聚体ODN与CAPL（S100A4）RNA的杂交，据报道该基因在乳腺癌和骨肉瘤等转移性癌症中过表达。将磷酸二酯ODN GX-1（反义）和两个对照序列（随机排列和随机）与双功能螯合剂S-苯甲酰基-巯基乙酰三甘氨酸（S-苯甲酰基-MAG3）偶联并用99锝标记。放射性标记的ODN在C18微型柱上纯化，并在反相高效液相色谱系统上进行表征。放射化学纯度>90%，产物在水性介质中稳定>6小时。使用聚丙烯酰胺凝胶电泳（PAGE）研究了未标记、32磷标记和99锝标记的ODN与转录RNA的杂交特性。证明了GX-1与转录RNA的直接杂交。未标记的ODN比转录RNA过量50倍会导致RNA酶H激活使RNA几乎完全消耗。在放射性标记（32磷和99锝）的ODN与RNA按1:1比例时，在PAGE系统中仅发现放射性标记的GX-1与RNA杂交。放射性标记的对照ODN未显示杂交迹象。本研究表明，ODN的3'-99锝标记不会干扰ODN在溶液中的杂交特性，使得99锝标记成为反义技术在成像领域未来发展的一种有吸引力的方法。
