Novel Separation and Assay Methods for Nucleic Acids

Singhal, Ram

Abstract

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.