TRITIUM LABELLING OF DNA & RNA BY CHEMICAL & ENZYMATIC SYNTHESIS

核酸作为一种潜在的生物技术， 治疗剂，例如作为反义剂、适体、核酶 和用于基因治疗的双链体。 有很多具体的 由RNA、DNA或经修饰的核酸制备的试剂的应用 个核苷酸 一个具体的目标是开发那些DNA适体， 在癌症或血栓形成中具有重要作用靶向肽 并且允许使用正电子发射断层扫描进行非侵入性成像 （PET）。 关于稳定性的几个关键问题， 细胞分布必须得到充分解决，这使得 需要氚化适体。 氚标记2'-溴-d-ATP（10 mg） 在磷酸盐缓冲液（pH 8.6）中使用T2和PdO以产生200 mCi的 比活度26 Ci/mmole的2 - 3H-dATP。 这种氚化dATP是 用于使用33个碱基的模板产生16聚体凝血酶适体， DNA聚合酶的存在。 经过碱解和水解 分离出670_Ci的3H适体。 凝胶电泳和3H NMR 验证了氚标记的16-mer适体的纯度。 结合研究 已经表明，3H-16-mer类似物对凝血酶的亲和力是 几乎与原始适体的测量值相同。 稳定性 在分离的兔血液中检测了血液中的3H-适体 利用苯酚/氯仿萃取和离子交换HPLC。 的 适体在血液中降解，生物半衰期为 在15 - 30分钟之间。 HPLC保留模式表明， 降解发生在3 ′-和5 ′-末端。 虽然这 降解过程相对较快，需要 临床使用的短寿命PET同位素（例如11C），快速血液 清除可以增强血管中血栓的成像。 是 还可以想象，与凝血酶结合的适体在 vivo. 目前正在研究这种可能性， 去内皮化的主动脉兔模型，3H-适体，和 色谱法和放射自显影法来测定 适体对凝血酶的特异性和适体的稳定性 与血凝块中的凝血酶结合的适体。

Nucleic acids have evoked extensive interest as potential therapeutical agents, e.g. as antisense agents, aptamers, ribozymes and duplexes for gene therapy. There are a wide range of specific application of such agents which are made from RNA, DNA or modified nucleotides. One specific goal is to develop those DNA aptamers which target peptides that have significant roles in cancer or thrombosis and which allow non-invasive imaging with Positron Emission Tomography (PET). Several crucial questions regarding stability, and tissues or cellular distribution must be fully addressed, which makes the tritiated aptamer desirable. 2'-bromo-d-ATP (10 mg) was tritiated using T2 and PdO in phosphate buffer (pH 8.6) to yield 200 mCi of 2-3H-dATP of specific activity 26 Ci/mmole. This tritiated dATP was used to produce a 16-mer thrombin aptamer using a 33 base template in the presence of DNA polymerase. After desalting and base hydrolysis 670 _Ci of 3H aptamer was isolated. Gel electrophoresis and 3H NMR verified the purity of the tritiated 16-mer aptamer. Binding studies have indicated that the affinity of 3H-16-mer analog for thrombin is nearly the same as measured for the original aptamer. Stability of the 3H-aptamer in blood has been examined in isolated rabbit blood utilizing phenol/chloroform extractions and ion exchange HPLC. The aptamer was degraded in the blood, with a biological half-life of between 15-30 minutes. The HPLC retention patterns indicate that degradation occurs at both the 3'- and 5'-termini. Though this degradative process is relatively rapid, and would require a short-lived PET isotope for clinical use (e.g. 11C), the rapid blood clearance could enhance imaging of thrombi in blood vessels. It is also conceivable that aptamer bound to thrombin will be more stable in vivo. This possibility is currently being studied with a deendothelialized aorta rabbit model, the 3H-aptamer, and chromatographic and autoradiographic methods to determine both the specificity of the aptamer to thrombin and the stability of the aptamer bound to thrombin in blood clots.