DESIGN AND SYNTHESIS OF NUCLEOTIDE ANALOGUES

（申请人的描述）该计划项目的一个重要目标是 开发高灵敏度聚合酶链反应/限制性酶切 核酸内切酶/连接酶检测反应（PCR/RE/LDR）方法鉴定 存在过量正常DNA时的特异性突变（灵敏度为1 在100，000 - 1，000，000个细胞中）。为此，一类新的核苷酸 本文中称为“转化体”的类似物被设计成提供 是指将野生型序列转化为含有 限制性内切酶识别位点。收敛定义为 具有碱基修饰或置换的核苷类似物， 允许它们在杂交中与一个或多个天然碱基配对 步骤（“读取”），并且还用作 DNA复制反应（“写”）。一个成功的融合将配对 当与靶退火时，一个或多个天然碱基， 标签聚合酶（读）的有效延伸，也作为一个 用于掺入另一个碱基的模板，标签聚合酶复制 包含引物的链（写入）。 为了执行12种可能的碱基转换，已知7种碱基转换是已知的。 脱氧核苷类似物（Q1、Q2、Q4-Q8）和13种新设计的， 修饰的脱氧核糖核苷（Q3，Q9-20）将被研究。 具体 目标包括：脱氧核糖核苷Q3，Q9-20的合成路线 和他们的性格。([1]1H和[13] C NMR，质谱， 吸收光谱和元素分析; 5 '- 转化物的二甲氧基三苯甲基（DMT）保护的衍生物将是 制备并转化为3 '-亚磷酰胺和3'-连接的CPG固体 用于掺入项目1和3的寡核苷酸的载体。的 将确定修饰的核苷酸对双链体结构的影响 通过测量和分析双链体组的螺旋-卷曲过渡 含有与每个天然核巧相对的每个Q核巧， 核苷具有骨架（磷酸盐和糖）的引物的合成 将探索在3 '端或接近3'端的修饰用于：（1）防止 在PCR反应中通过校正聚合酶切割3 ′-convertide （项目1）和（2）通过以下方法提高连接酶反应的保真度： 连接酶-DNA复合物的去稳定化（项目3）。

(Applicant's Description) An important goal of the program project is the development of a high sensitivity polymerase chain reaction/restriction endonuclease/ligase detection reaction (PCR/RE/LDR) method to identify specific mutations in the presence of excess normal DNA (Sensitivity of 1 in 100,000 - 1,000,000 cells). To this end, a new class of nucleotide analogues herein termed "convertides", have been designed to provide a means to convert a wild-type sequence into one which contains a restriction endonuclease recognition site. Convertides are defined as nucleoside analogues that have base modifications or replacements which allow them to pair to one or more of the natural bases in hybridization step ("read"), and also to function as a template for another base in a DNA replication reaction ("write"). A successful convertide will pair to one or more natural bases when annealing to a target, allowing for efficient extension by Tag polymerase (read), and also functions as a template for incorporation of another base the tag polymerase copies the primer-containing strand (write). For performing the 12 possible base conversions, 7 known deoxyribonucleoside analogues (Q1, Q2, Q4-Q8) and 13 newly-designed, modified deoxyribonucleosides (Q3, Q9-20) will be investigated. Specific objectives include: Synthetic routes to deoxyribonucleosides Q3, Q9-20 and their characterization. ([1] H and [13] C NMR, mass spectrometry, absorption spectroscopy, and elemental analysis; Synthesis of 5'- Dimethoxytrityl (DMT)-protected derivatives of the convertides will be prepared and transformed to 3'-phophoramidites and 3'-linked CPG solid supports for incorporation into oligonucleotides for Projects 1 and 3. The effect of the modified nucleotides on duplex structure will be determined by measuring and analyzing the helix-coil transition of sets of duplexes containing each of the Q nucleosides opposite each of the natural nucleosides. Synthesis of primers with backbone (phosphate and sugar) modifications at or near the 3'-end will be explored for: (1) preventing cleavage of 3'-convertide by proofreading polymerases in PCR reactions (Project 1) and (2) improving fidelity of ligase reactions by destabilization of ligase-DNA complex (Project 3).