TRITIUM LABELLING OF DNA & RNA BY CHEMICAL & ENZYMATIC SYNTHESIS

核酸作为一种潜在的物质引起了人们的广泛兴趣 治疗药物，如反义药物、适配子、核酶 以及用于基因治疗的双链基因。有各种各样的具体的 核糖核酸、脱氧核糖核酸或经修饰的此类试剂的应用 核苷酸。一个具体的目标是开发那些能够 在癌症或血栓形成中有重要作用的靶标多肽 并允许使用正电子发射断层扫描进行非侵入性成像 (宠物)。关于稳定性和组织的几个关键问题 蜂窝分布必须得到充分解决，这使得 理想的氚适体。2‘-溴-d-三磷酸腺苷(10 Mg)进行氚滴定 在磷酸盐缓冲液(pH 8.6)中用T2和PDO得到200 mCI的 比活度为26cI/mMole的2-3H-dATP。这种氚化合成的dATP是 用于使用33个碱基模板生产16-聚凝血酶适配子 DNA聚合酶的存在。脱盐和碱水解后 分离得到了~3H适配子的670Ci。凝胶电泳法和~3H-核磁共振 验证了氚16-聚体适配子的纯度。约束性研究 已经表明，~3H-16-聚体类似物对凝血酶的亲和力是 与原始适配子的测量结果几乎相同。的稳定性 在分离的兔血中检测了血液中的~3H-适配子 利用苯酚/氯仿萃取物和离子交换高效液相色谱法。这个 适体在血液中被降解，生物半衰期为 15-30分钟之间。高效液相色谱保留图谱表明 降解发生在3‘-和5’-末端。虽然这件事 降解过程相对较快，需要 临床使用的短寿命PET同位素(如11C)，快速血液 清除可增强血管内血栓的成像。它是 同样可以想象的是，与凝血酶结合的适体在 活着。目前正在研究这种可能性， 去内皮化兔主动脉模型、~3H-适配子和 用层析法和放射自显影法测定 核酸适体对凝血酶的特异性及其稳定性 适体与血栓中的凝血酶结合。

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.