A semi-synthetic organism that stores and retrieves increased genetic information

一种存储和检索更多遗传信息的半合成生物体

• 批准号: 9469534
• 负责人: Floyd E. Romesberg
• 金额: $ 72.21万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9469534
• 关键词: Amino Acids; Base Pairing; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; DNA; Development; Engineering; Escherichia coli; Evolution; Foundations; Funding; Genes; Genetic; Genetic Transcription; Genome; Genomics; Health; Human; Hydrogen Bonding; Hydrophobicity; Instruction; Letters; Life; Mediating; Medicine; Messenger RNA; Nucleotides; Organism; Production; Proteins; RNA; Therapeutic; Transfer RNA; Triplet Multiple Birth; United States National Institutes of Health; Work; base; genetic information; hydrophilicity; improved; novel; nuclease; promoter; public health relevance; therapeutic protein; translational approach; tripolyphosphate; unnatural amino acids; uptake

 DESCRIPTION (provided by applicant): Organisms are defined by the information encoded in their genomes, and since the evolution of life as we know it, this information has been encoded using a four-letter genetic alphabet, made possible by the selective pairing of (d)G with (d)C and (d)A with dT or U. The creation of a third, unnatural base pair (UBP) would have profound implications for our understanding of what life is and how it may have evolved, and could serve as the foundation of a semi-synthetic organism (SSO), a living cell that stores information beyond that of the natural genetic alphabet and retrieves it in the form of proteins containing unnatural amino acids. This has great potential to improve human health, as proteins now constitute an important class of therapeutics, but their utility is currently restricted by the limted physicochemical diversity of the twenty natural amino acids. Since 1999, our NIH-funded work has focused on the development of a UBP. Our strategy is based on the use of hydrophobic and packing forces, as opposed to Watson-Crick-like hydrogen bonds, to optimize UBP formation, as we have found that such forces are strong and disfavor mispairing with the natural, more hydrophilic nucleotides. This effort reached major milestones in 2009 with our discovery of the UBP formed between dNaM and d5SICS, and in 2014 with our engineering of an E. coli-based SSO that stably harbors this UBP in its DNA. In the past year, we have continued to optimize the SSO, including the optimization and genomic integration of the gene encoding the nucleotide triphosphate transporter from Phaeodactylum tricornutum (PtNTT2), which makes import of dNaMTP and d5SICSTP into the cell possible. With the optimized PtNTT2 now under the control of a constitutive promoter, the SSO is more healthy and always competent for unnatural triphosphate uptake. While we have discovered that replication of the UBP proceeds with a sequence bias, we have already made progress towards eliminating the bias by continuing to optimize the UBP, and by introducing an error-correction mechanism mediated by CRISPR-associated protein-9 nuclease (Cas9). We have also demonstrated that DNA containing the UBP may be transcribed within the SSO into RNA containing unnatural nucleotides. Although continued exploration and optimization is still required, the major challenges of creating the first form of life that stably harbors and retrieves information beyond that encoded by the natural genetic alphabet have been identified. These include the optimization of replication to eliminate the observed sequence bias, the optimization of transcription, including the transcription of mRNAs and tRNAs, and lastly, the demonstration of efficient translation, and strategies toward overcoming these challenges are described. If successful, our efforts will yield the first form of life that faithfully stores and retrieves infomation beyond that encoded by the natural genetic alphabet, and will result in a general platform for the production of diverse, therapeutic proteins that could revolutionize medicine.

 描述(申请人提供)：生物体是由其基因组中编码的信息定义的，自我们所知的生命进化以来，该信息已使用四个字母的遗传字母表进行编码，这是通过选择性地将(D)G与(D)C和(D)A与DT或U配对而实现的。第三个非自然碱基对(UBP)的创建将对我们理解生命是什么以及它可能如何进化具有深远的影响，并可作为半合成有机体(SSO)的基础，一种活细胞，它存储自然遗传字母表之外的信息，并以含有非天然氨基酸的蛋白质的形式检索这些信息。这对改善人类健康具有巨大的潜力，因为蛋白质现在构成了一类重要的治疗药物，但其用途目前受到20种天然氨基酸有限的物理化学多样性的限制。自1999年以来，我们由NIH资助的工作一直集中在UBP的发展上。我们的策略是基于疏水和填充力的使用，而不是Watson-Crick类氢键，以优化UBP的形成，因为我们发现这种力很强，不利于与天然的、更亲水的核苷酸错配。这一努力在2009年达到了重大里程碑，我们发现了在dNaM和d5SICS之间形成的UBP，并在2014年设计了一种基于大肠杆菌的SSO，它在DNA中稳定地含有这种UBP。在过去的一年里，我们继续优化SSO，包括编码三角褐指藻核苷酸三磷酸转运蛋白(PtNTT2)的基因的优化和基因组整合，这使得dNaMTP和d5SICSTP的导入成为可能。优化后的PtNTT2现在处于构成启动子的控制下，SSO更健康，始终能够吸收非自然的三磷酸盐。虽然我们已经发现UBP的复制是以序列偏差进行的，但我们已经通过继续优化UBP和引入由CRISPR相关蛋白-9核酸酶(Cas9)介导的纠错机制来消除这种偏差。我们还证明了含有UBP的DNA可以在SSO内转录成含有非天然核苷酸的RNA。尽管仍然需要继续探索和优化，但已经确定了创造第一种生命形式的主要挑战，这种生命形式稳定地储存和检索自然遗传字母表所编码的信息之外的信息。其中包括优化复制以消除观察到的序列偏差，优化转录，包括mRNAs和tRNAs的转录，最后展示有效的翻译，并描述了克服这些挑战的策略。如果成功，我们的努力将产生第一种形式的生命，它忠实地存储和检索自然遗传字母表所编码的信息之外的信息，并将产生一个生产各种治疗性蛋白质的通用平台，这可能会给医学带来革命性的变化。

项目成果

Expansion of the genetic code via expansion of the genetic alphabet.

• DOI: 10.1016/j.cbpa.2018.08.009
• 发表时间: 2018-10
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8
• 作者: Dien VT;Morris SE;Karadeema RJ;Romesberg FE
• 通讯作者: Romesberg FE

Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons.

• DOI: 10.1021/jacs.9b09080
• 发表时间: 2019-12
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Anne Xiao-Zhou Zhou-Anne-Xiao-Zhou-Zhou-13769862;Kai Sheng;Aaron W Feldman;F. Romesberg

A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria.

• DOI: 10.1021/jacs.7b11404
• 发表时间: 2018-01-31
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Feldman AW;Fischer EC;Ledbetter MP;Liao JY;Chaput JC;Romesberg FE
• 通讯作者: Romesberg FE