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 这项集中的技术研究和发展建议迫切需要新工具 对长核酸底物的DNA和RNA核酶进行体外位点特异性修饰。这 DNA和RNA的生物化学，生物物理学和生物学受到特定核碱基的影响很大 修改。但是，对这些修饰的研究通常受到合成修改的能力的限制 核酸。因此，需要创新的新技术来规避现有的局限性 化学和酶修饰方法。 dnazymes（脱氧核酶）是具有酶促功能的人工DNA序列，类似于蛋白质 酶作为氨基酸的催化序列。尽管dnazymes本质上是未知的，但它们可能是 通过从随机DNA序列库开始并进行体外选择，在实验室中识别从头识别。 对于所需的酶活性。 Dnazymes在操作上很容易获得和使用：它们很容易 通过固相合成在商业上制备，它们很容易在简单的水性中修改其底物 与标准缓冲液和盐一起孵育条件。已经确定了dnazymes的范围 催化活性，但大多数dnazymes的应用仅限于RNA的两个长期反应 裂解和血素依赖性过氧化，与DNA和RNA核酶修饰无关。 在拟议的研究中，将确定DNAZYMES针对特定于网站特定体外的质量新方法 通过N-酰基化或（通过还原胺化）N4的N-氨基化修饰DNA和RNA核酶的修饰 胞嘧啶，鸟嘌呤的N2和腺嘌呤的N6。而不是依靠天然蛋白质酶，要么如发现 本质上或通过定向进化而进化 自然进化史将通过体外选择来识别。 提出了三个具体目标。在AIM 1中，体外选择将用于鉴定n-的dnazymes DNA和RNA底物中C，G和A核碱胺的酰化。在平行AIM 2中，体外选择将 用于通过还原C，G和核碱胺在DNA中的还原性胺化来鉴定N-烷基化。 和RNA底物。在这两个目的中，亲电反应伴侣将是寡核苷酸或 一个小分子，作为AIM 1中的酰基供体的氟化芳基酯，作为芳香或脂肪族 醛或激活的亚胺变体，用于AIM 2中的还原胺化伴侣。在AIM 3中，Dnazymes 从目标1和2的底物序列范围中，将从目标1和2进行重新选择。 为了提高功能，以促进实际用作特定网站DNA和RNA的创新新技术 核酶修饰。 通过拟议的努力结束，我们将确定Dnazymes是一个重要的新事物 通过N-酰基化和N-烷基化修饰体外位点特异性DNA和RNA核酶修饰的技术。