DNAzymes for Site-Specific DNA and RNA Nucleobase Modification

用于位点特异性 DNA 和 RNA 核碱基修饰的 DNAzyme

• 批准号: 10630686
• 负责人: Scott K Silverman
• 金额: $ 29.17万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630686
• 关键词: Acylation; Address; Adenine; Aldehydes; Alkylation; Alkynes; Amination; Amines; Amino Acid Sequence; Azides; Base Pairing; Binding; Biochemical; Biochemistry; Biological; Biology; Biophysics; Buffers; Catalysis; Catalytic DNA; Chemicals; Cytosine; DNA; DNA Modification Process; DNA Sequence; Development; Directed Molecular Evolution; Enzymes; Esters; Goals; Guanine; Hemin; Hydrolysis; Imines; In Vitro; Incubated; Individual; Laboratories; Length; Messenger RNA; Modification; N acylation; Nature; Nucleic Acids; Nucleotides; Oligonucleotides; Peptides; Population; Positioning Attribute; Procedures; Proteins; Publications; RNA; RNA Ligase (ATP); Randomized; Reaction; Recording of previous events; Reporting; S phase; Salts; Series; Site; Solid; Specificity; Technology; Variant; Work; amino group; aqueous; experimental study; functional group; functional improvement; innovation; new technology; novel strategies; nucleobase; peroxidation; pressure; small molecule; technology research and development; tool

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.

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核碱基进行体外位点特异性修饰。的 DNA和RNA的生物化学、生物物理学和生物学都受到特定核碱基的严重影响 修改.然而，对这些修饰的研究通常受到合成修饰的化合物的能力的限制。 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。 脱氧核酶（deoxyribozymes）是具有酶功能的人工DNA序列，类似于蛋白质 酶作为氨基酸的催化序列。尽管DNA酶本质上是未知的，但它们可以是 通过从随机DNA序列库开始并进行体外选择， 以获得所需的酶活性。DNA酶在操作上易于获得和用途：它们易于 商业上通过固相合成制备，并且它们容易在简单的水溶液中修饰它们的底物， 孵育条件与标准缓冲液和盐。DNA酶已被鉴定用于越来越多的 催化活性，但DNA酶的大多数应用仅限于RNA的两个长期已知的反应 切割和氯化血红素依赖的过氧化，这与DNA和RNA核碱基修饰无关。 在拟议的研究中，DNA酶将被确定为一种定性的新方法，以位点特异性体外 DNA和RNA核碱基的修饰，通过N-酰化或（通过还原胺化）N4位的N-烷基化 胞嘧啶的N2，鸟嘌呤的N2，和腺嘌呤的N6。而不是依赖于天然蛋白酶，无论是发现 在自然界中或通过定向进化而进化的，其序列不受 自然进化史将通过体外选择来鉴定。 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-脱氧核糖核酸酶。 DNA和RNA底物中C、G和A核碱基胺的酰化。在平行目标2中，体外选择将 用于通过DNA中C、G和A核碱基胺的还原胺化来鉴定用于N-烷基化的DNA酶 和RNA底物。在这两个目标中，亲电反应配偶体将是寡核苷酸或 小分子，作为目标1中酰基供体的可调氟化芳基酯，和作为芳族或脂族 醛或其活化的亚胺变体，用于目标2中的还原胺化配偶体。在目标3中，DNA酶 根据目标1和目标2，将根据其基质序列范围进行表征，并通过重新选择进行细化 为改善功能，以促进实际使用，作为一种创新的新技术，为位点特异性的DNA和RNA 核碱基修饰 通过所提出的努力的结论，我们将建立DNA酶作为一个重要的新的 通过N-酰化和N-烷基化体外位点特异性DNA和RNA核碱基修饰技术。