DNAzymes for Site-Specific DNA and RNA Nucleobase Modification

项目摘要 这一重点技术研究和开发提案解决了对新工具的迫切需求 对长核酸底物的DNA和RNA碱基进行体外定点修饰。这个 Dna和rna的生物化学、生物物理学和生物学都受到特定碱基的严重影响。 修改。然而，对这些修饰的研究往往受到合成修饰的能力的限制 核酸。因此，需要创新的新技术来规避现有技术的局限性 化学和酶改性方法。 DNAzyme(脱氧核酶)是具有酶功能的人造DNA序列，类似于蛋白质 酶作为氨基酸的催化序列。虽然DNAzyme在自然界中是未知的，但它们可以 通过从随机DNA序列库开始并进行体外选择来在实验室进行重新鉴定 以获得所需的酶活性。DNAzyme在操作上很容易获得和使用：它们很容易 商业上通过固相合成制备，它们很容易在简单的水溶液中修饰其底物 标准缓冲液和盐的孵化条件。DNAzyme已被鉴定为越来越多的 催化活性，但大多数DNAzyme的应用仅限于已知的两个RNA反应 裂解和氯化血红素依赖的过氧化，与DNA和RNA碱基修饰无关。 在拟议的研究中，DNAzyme将被确定为一种在体外定位的定性新方法 DNA和RNA碱基的修饰，通过N-酰化或(通过还原胺化)N-烷基化 胞嘧啶，鸟嘌呤N_2，腺嘌呤N_6。而不是依赖于天然的蛋白质酶，无论是发现的 在自然界中或通过定向进化进化而来的全新的DNA酶，其序列不受 自然进化史将通过体外选择来确定。 提出了三个具体目标。在目标1中，将使用体外选择来鉴定N-DNA酶。 DNA和RNA底物中C、G和A核碱胺的酰化反应。在平行的目标2中，体外选择将 利用DNA中C、G、A核碱基胺的还原胺化反应鉴定N-烷基化脱氧核酶 和RNA底物。在这两个目的中，亲电反应伙伴将是寡核苷酸或 一种小分子，如目标1中的酰基供体的可调节的氟化芳基酯，以及芳香族或脂肪族 醛或其活化亚胺变体，用于目标2中的还原胺化伙伴。在目标3中，脱氧核酶 目标1和目标2的特征在于它们的底物序列范围，并通过重新选择来提炼 为了改善功能，促进实际使用，作为一种创新的特定部位DNA和RNA的新技术 碱基修饰。 到提议的努力结束时，我们将把DNAzyme确立为一种重要的新的 通过N-酰化和N-烷基化对DNA和RNA进行体外定点修饰的技术。

Project Summary This Focused Technology Research and Development proposal addresses the urgent need for new tools to perform in vitro site-specific modification of DNA and RNA nucleobases of long nucleic acid substrates. The biochemistry, biophysics, and biology of DNA and RNA are heavily influenced by particular nucleobase modifications. However, studies of these modifications are often limited by the ability to synthesize the modified nucleic acids. Therefore, innovative new technologies are required to circumvent the limitations of the existing chemical and enzymatic modification approaches. DNAzymes (deoxyribozymes) are artificial DNA sequences with enzymatic function, analogous to protein enzymes as catalytic sequences of amino acids. Although DNAzymes are unknown in nature, they can be identified de novo in the laboratory by starting with random DNA sequence pools and performing in vitro selection for the desired enzymatic activity. DNAzymes are operationally simple to obtain and use: they are readily prepared commercially by solid-phase synthesis, and they easily modify their substrates in simple aqueous incubation conditions with standard buffers and salts. DNAzymes have been identified for a growing range of catalytic activities, but most applications of DNAzymes are limited to the two long-known reactions of RNA cleavage and hemin-dependent peroxidation, which are unrelated to DNA and RNA nucleobase modification. In the proposed studies, DNAzymes will be identified for a qualitatitvely new approach to site-specific in vitro modification of DNA and RNA nucleobases, by either N-acylation or (via reductive amination) N-alkylation at N4 of cytosine, N2 of guanine, and N6 of adenine. Rather than relying on natural protein enzymes, either as found in nature or evolved by directed evolution, entirely new DNAzymes whose sequences are unconstrained by natural evolutionary history will be identified by in vitro selection. Three specific aims are proposed. In Aim 1, in vitro selection will be used to identify DNAzymes for N- acylation of C, G, and A nucleobase amines in DNA and RNA substrates. In parallel Aim 2, in vitro selection will be used to identify DNAzymes for N-alkylation by reductive amination of C, G, and A nucleobase amines in DNA and RNA substrates. In both of these aims, the electrophilic reaction partner will be either an oligonucleotide or a small molecule, as a tunably fluorinated aryl ester for the acyl donor in Aim 1, and as an aromatic or aliphatic aldehyde, or activated imine variant thereof, for the reductive amination partner in Aim 2. In Aim 3, the DNAzymes from Aims 1 and 2 will be characterized with regard to their substrate sequence scope and refined by reselection for improved function, to facilitate practical use as an innovative new technology for site-specific DNA and RNA nucleobase modification. By the conclusion of the proposed efforts, we will have established DNAzymes as an important new technology for in vitro site-specific DNA and RNA nucleobase modification by N-acylation and N-alkylation.