DESIGN AND SYNTHESIS OF NUCLEOTIDE ANALOGUES

(申请者描述)该项目的一个重要目标是 高灵敏度聚合酶链式反应/限制性内切酶的建立 核酸内切酶/连接酶检测反应(PCR/RE/LDR)方法鉴定 在存在过量正常DNA的情况下发生特定突变(敏感度为1 在100,000-1,000,000个单元中)。为此，一类新的核苷酸 类似物在这里被称为“汇聚”，其设计目的是提供一种 将野生型序列转换为包含 限制性内切酶识别位点。汇聚的定义为 具有碱基修饰或取代的核苷类似物 允许它们在杂交中与一个或多个天然碱基配对 步骤(“读”)，并且还用作一个模板，用于 DNA复制反应(“写”)。成功的转换将配对到 一个或多个天然碱基，当对目标进行退火时，允许 标签聚合酶(Read)的高效延伸，也作为一种 用于结合另一个碱基的模板标签聚合酶复制 含有底物的链(写)。 对于执行12种可能的基本转换，7个已知 脱氧核糖核苷类似物(Q1、Q2、Q4-Q8)和13个新设计、 将研究修饰的脱氧核糖核苷(Q3、Q9-20)。特定的 目标包括：脱氧核糖核苷Q3、Q9-20的合成路线 以及他们的性格特征。([1]H和[13]C核磁共振，质谱学， 吸收光谱分析和元素分析.5‘-的合成 受二甲氧基三苯基(DMT)保护的转化率的衍生物将是 3‘-亚磷酰胺和3’-连接CPG固体的制备及转化 支持纳入项目1和3的寡核苷酸。 修饰的核苷酸对双链结构的影响将被确定 通过测量和分析多组双链的螺旋-线圈转变 含有每一个Q核苷，与每个天然的 核苷。含主链(磷酸和糖)的底物的合成 将探索在3‘端或其附近的修改：(1)防止 PCR反应中聚合酶对3‘-转化子的切割作用 (项目1)和(2)通过以下方式提高连接酶反应的保真度 连接酶-DNA复合体的不稳定性(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).