Elucidating sequence-function relationships in a template-independent polymerase to enable novel DNA recording applications

IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology and Bioengineering Pub Date : 2024-09-14 DOI:10.1002/bit.28838
Marija Milisavljevic, Teresa Rojas Rodriguez, Keith E. J. Tyo
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Abstract

Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3′ end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships.

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阐明独立于模板的聚合酶的序列功能关系,实现新型 DNA 记录应用
利用 DNA 作为信息存储和信号分子记录的高密度存储介质,在生物技术领域引起了越来越多的关注。最近,利用不依赖模板的 DNA 聚合酶--末端脱氧核苷酸转移酶(TdT)的活性,在酶法 DNA 合成、DNA 数字数据存储和基于 DNA 的分子记录方面取得了进展。TdT 将脱氧核苷酸添加到单链 DNA 的 3′端,生成单链 DNA 的随机序列。TdT 的酶活性可使用多种二价阳离子,并随着反应环境的变化而改变脱氧核苷酸的掺入频率。然而,我们对这些特性的序列-结构-功能关系了解有限,这反过来又限制了我们调节 TdT 以进一步推动基于 TdT 的工具的能力。迄今为止,大多数 TdT 文献探讨的是鼠类、牛类或人类 TdT 的活性;对 TdT 序列和结构的研究主要集中在严格保守的残基上,这些残基在功能上对 TdT 活性至关重要。在这里,我们通过调查 TdT 的天然多样性来探索非保守的 TdT 序列空间。我们描述了来自不同生物体的各种 TdT 同源物,并确定了赋予同源物之间 TdT 行为差异的几个 TdT 残基/区域。本研究中的观察结果可以为靶向 TdT 文库设计规则,并与筛选试验相结合,以调节 TdT 的特性。此外,这些数据还有助于指导对潜在相关残基的进一步研究。总之,我们描述了以前文献中没有研究过的 TdT 的特性,并为 TdT 序列与功能的关系提供了新的见解。
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来源期刊
Biotechnology and Bioengineering
Biotechnology and Bioengineering 工程技术-生物工程与应用微生物
CiteScore
7.90
自引率
5.30%
发文量
280
审稿时长
2.1 months
期刊介绍: Biotechnology & Bioengineering publishes Perspectives, Articles, Reviews, Mini-Reviews, and Communications to the Editor that embrace all aspects of biotechnology. These include: -Enzyme systems and their applications, including enzyme reactors, purification, and applied aspects of protein engineering -Animal-cell biotechnology, including media development -Applied aspects of cellular physiology, metabolism, and energetics -Biocatalysis and applied enzymology, including enzyme reactors, protein engineering, and nanobiotechnology -Biothermodynamics -Biofuels, including biomass and renewable resource engineering -Biomaterials, including delivery systems and materials for tissue engineering -Bioprocess engineering, including kinetics and modeling of biological systems, transport phenomena in bioreactors, bioreactor design, monitoring, and control -Biosensors and instrumentation -Computational and systems biology, including bioinformatics and genomic/proteomic studies -Environmental biotechnology, including biofilms, algal systems, and bioremediation -Metabolic and cellular engineering -Plant-cell biotechnology -Spectroscopic and other analytical techniques for biotechnological applications -Synthetic biology -Tissue engineering, stem-cell bioengineering, regenerative medicine, gene therapy and delivery systems The editors will consider papers for publication based on novelty, their immediate or future impact on biotechnological processes, and their contribution to the advancement of biochemical engineering science. Submission of papers dealing with routine aspects of bioprocessing, description of established equipment, and routine applications of established methodologies (e.g., control strategies, modeling, experimental methods) is discouraged. Theoretical papers will be judged based on the novelty of the approach and their potential impact, or on their novel capability to predict and elucidate experimental observations.
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