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NOVEL SEPARATION AND ASSAY METHODS FOR NUCLEIC ACIDS

核酸的新分离和测定方法

基本信息

批准号：
3438414
负责人：
RAM SINGHAL
金额：
$ 6.25万
依托单位：
WICHITA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-11-01 至 1988-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3438414
关键词：
affinity chromatography aging aminoacyl tRNA boron chemical structure function chromatography fluorescence high performance liquid chromatography molecular biology neoplasm /cancer nucleic acid chemical synthesis oligonucleotides physical separation transfer RNA

项目摘要

Specific transfer RNA (tRNA) is needed for studying the structure and function of tRNAs. Current isolation methods are cumbersome, time-consuming, and produce only a small amount of the material. Homogeneous oligodeoxynucleotides of predetermined sequence are needed for numerous molecular biology studies. Although they can now be synthesized easily, purification of the product in the amounts needed is a real problem. DNA methylation for control of gene expression in aging and cancer has been considered; however, information on the chemical basis of this notion is missing. The problem lies not as much with the resolution of mdC from dC (or mdA from dA), but with their presence in DNA in only trace amounts. To solve these problems, three specific research goals are proposed: (1) Development of a satisfactory boronate affinity matrix for a one-step purification of several important metabolites, especially specific tRNAs. (2) Study of the separation of tailor-made oligonucleotides by High performance liquid chromatography (Hplc). (3) Derivatization of mdC (and mdA) with an ultraviolet (uv) and fluorescent marker to enhance detection and assay of this important component in DNA analysis by Hplc. We recently synthesized a reversed-phase boronate matrix to resolve one specific aminoacyl-tRNA from other (as many as 19) nonacyl tRNAs in a one-step purification scheme. Although some of the results are very fruitful, this affinity matrix still fails to function in an acidic condition essential for stability of AA-tRNA bond. We propose introduction of a nitro group to the phenylamine ring carrying the reactive boronic acid. Nitration, in addition to countering the electron donating effect of the amino group, is expected to appreciably lower the pK of boronic acid, thus allowing complex formation with cis-diols at an acid pH. Synthesis and evaluation of a reversed-phase and silica-based matrices of several important compounds of clinical interest are proposed. Isolation and purification of a synthetic d(GATCGATCGATC) was examined on several Hplc matrices. The results indicate the product, after synthesis is a complex mixture. It calls for a careful study of different column matrices and Hplc parameters to obtain mg amounts of the pure material. We propose study of a dual-column chromatography procedure to achieve this goal. Our current study of mdC contents in DNA samples from different age groups of mice indicate that dmC contents indeed decrease with age. To extend this work and enhance sensitivity of DNA analysis, reaction of chloroacetaldehyde with mdC and dC (similarily with mdA and dA) is proposed. Establishment of fluorescent and uv parameters for the reaction products and study of DNA analysis by different Hplc columns are proposed.

需要特异性转移RNA（tRNA）来研究其结构， tRNA的功能。目前的隔离方法很麻烦，耗时，并且仅产生少量的材料。需要预定序列的同质寡脱氧核苷酸，许多分子生物学研究。虽然它们现在可以被合成容易地，纯化所需量的产物是真实的，问题. DNA甲基化在衰老和衰老过程中的基因表达调控癌症已经被考虑;然而，关于癌症的化学基础的信息这个概念是缺失的。问题不在于决议 mdC来自dC（或mdA来自dA），但它们在DNA中的存在仅限于微量针对这些问题，提出了三个具体的研究目标：（1）一步法制备硼酸盐亲和基质的研究纯化几种重要的代谢物，特别是特定的tRNA。 (2)高效液相色谱分离定制寡核苷酸的研究高效液相色谱法（Hplc）。 (3)mdC的衍生化（和 mdA）带有紫外线（uv）和荧光标记物以增强检测用高效液相色谱法测定了DNA分析中这一重要组分的含量。我们最近合成了一种反相硼酸盐基质来解决一个问题特异性氨酰-tRNA从其他（多达19个）非酰基tRNA中分离出来，一步纯化方案。虽然有些结果非常富有成效，这种亲和基质仍然不能在酸性环境中发挥作用。 AA-tRNA键稳定的必要条件。我们建议引入的硝基基团的苯胺环携带反应性硼酸硝化，除了抵消的电子供体效应的氨基，预期可明显降低硼酸的pK，从而允许在酸性pH下与顺式二醇形成络合物。和评价几种化合物的反相和基于二氧化硅的基质提出了临床上感兴趣的重要化合物。分离与在几种HPLC上检查合成d（GATCGATCGATC）的纯化矩阵结果表明，合成后的产物为配合物混合物. 它要求仔细研究不同的列矩阵， HPLC参数以获得mg量的纯物质。我们提出双柱层析程序的研究，以实现这一目标。我们不同年龄组儿童DNA中mdC含量的研究现状小鼠表明dmC含量确实随着年龄的增长而降低。延长本工作和提高灵敏度的DNA分析，反应氯乙醛与mdC和dC（类似于mdA和dA）是提出了反应的荧光和紫外参数的建立产品和研究的DNA分析不同的HPLC柱。