Full-length sequencing of individual RNAs from heterogeneous samples

对异质样品中的单个 RNA 进行全长测序

• 批准号: 10482321
• 负责人: William S Agnew
• 金额: $ 39.8万
• 依托单位: PHAENO INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10482321
• 关键词: 2019-nCoV; AIDS/HIV problem; Address; Alzheimer' s Disease; Biological; COVID-19; Cancer Diagnostics; Cells; Chemistry; Circular DNA; Communicable Diseases; Complementary DNA; Complex; Coronavirus; Custom; DNA; DNA Library; DNA Primers; DNA Repair; DNA biosynthesis; DNA sequencing; Directed Molecular Evolution; Disease; Enzymatic Biochemistry; Enzymes; Feasibility Studies; Fee-for-Service Plans; Foundations; Genes; Genetic Transcription; Genomics; Goals; HIV; High-Throughput DNA Sequencing; Homo; Human; Immunotherapy; In Vitro; Individual; Industry Standard; Intellectual Property; Introns; Investments; Legal patent; Length; Libraries; Licensing; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Methods; Morphologic artifacts; Muscular Dystrophies; Nucleic Acids; Performance; Phase; Polymerase; Positioning Attribute; Preparation; Proteins; Protocols documentation; RNA; RNA Splicing; RNA Viruses; RNA primers; RNA replication; RNA-Directed DNA Polymerase; Reading; Reagent; Recovery; Reproducibility; Retrotransposon; Retroviridae; Reverse Transcription; Ribonuclease H; Rights; Sampling; Secure; Services; Single-Stranded DNA; Site-Directed Mutagenesis; Source; Technology; Testing; Translations; Universities; Validation; Variant; Viral Genome; base; comparative; computerized data processing; drug discovery; improved; instrument; next generation; novel; novel strategies; novel therapeutics; personalized diagnostics; phase 2 study; physical separation; precision medicine; prevent; single cell sequencing; single molecule; single-cell RNA sequencing; therapeutic gene; transcriptome; transcriptome sequencing

Summary Despite large investments in nucleic acid technology, the ability to sequence large numbers of full-length individual RNAs, from complex samples, with highest accuracy, has remained out of reach. Pheno introduces an advance for large scale Next Generation DNA Sequencing (NGS) sequencing of effectively unlimited numbers of individual RNA molecules from heterogeneous mixtures. We seek to prove that these methods can be implemented with the scale and precision to justify translation into commercially viable products and services. The aims of this proposal address two key steps that determine scale and accuracy at which the technology can be applied, exploiting recent discoveries in nucleic acid enzymology. Aim #1 focuses on reverse transcription, prompted by creation, with in vitro directed evolution, of a highly accurate, proof-reading reverse transcriptase, with high processivity and devoid of integral RNase H activity and other sources of RT artefacts. This advance over even the best retroviral and enzymes derived from retrotransposons or group-ii introns promises accurate replication of even the longest RNA viral genomes. Conventional retroviral enzymes, for example, even modified by site directed mutagenesis, are prone to at least 11 transcriptional artefacts in addition to simple base-reading errors. Aim #2 exploits a recently discovered class of primer-polymerases used in DNA repair. A key step in our sequencing chemistry exploits topological advantages of homo-concatamers of tagged cDNAs produced by Rolling Circle Amplification (RCA) of circularized single-stranded templates. The lowest scale at which the technology can be applied is limited by conventional RCA protocols that depend on exogenous random DNA primers. and generate artifactual sequences with samples of small size. New protocols use a PrimPol polymerase to synthesize RNA primers directly from the template, preventing de novo artefacts and simultaneously improving amplification by ~ 5 orders of magnitude. This presents the exciting possibility of applying the technology to samples below the scale of a single-cell transcriptome. Collectively these studies will seek to set new industry standards for RNA sequencing. This could help accelerate a wide range of precision medicine, viz. precision cancer diagnostics, immunotherapy; therapeutic gene editing; new drug discovery and validation. The technology could provide transformational advances in battling infectious diseases, including HIV/AIDS and SARS-CoV-2-mediated COVID 19.

概括 尽管对核酸技术进行了大量投资，但对大量全长序列进行测序的能力 从复杂样本中提取的单个 RNA 的准确性仍然遥不可及。菲诺介绍 大规模下一代 DNA 测序 (NGS) 测序的进步，有效数量无限 来自异质混合物的单个 RNA 分子。我们试图证明这些方法可以 实施的规模和精度足以证明转化为商业上可行的产品和服务是合理的。 该提案的目标是确定技术可实现的规模和准确性的两个关键步骤 利用核酸酶学的最新发现进行应用。目标#1专注于逆转录， 通过体外定向进化创造出一种高度准确、校对的逆转录酶， 具有高持续合成能力，并且没有完整的 RNase H 活性和其他 RT 假象来源。这一进展 即使是最好的逆转录病毒和源自逆转录转座子或第二组内含子的酶也能保证准确 甚至最长的RNA病毒基因组的复制。例如，传统的逆转录病毒酶甚至经过修饰 通过定点诱变，除了简单的碱基读取外，还容易产生至少 11 种转录产物 错误。目标 #2 利用最近发现的一类用于 DNA 修复的引物聚合酶。我们的关键一步 测序化学利用了标记 cDNA 同源联体的拓扑优势 环化单链模板的滚环扩增 (RCA)。最低规模 技术的应用受到依赖于外源随机 DNA 的传统 RCA 方案的限制 引物。并用小样本生成人工序列。新协议使用 PrimPol 聚合酶直接从模板合成 RNA 引物，防止从头假象和 同时将放大倍数提高约 5 个数量级。这提供了令人兴奋的可能性 将该技术应用于单细胞转录组规模以下的样本。总的来说，这些研究将 寻求为 RNA 测序制定新的行业标准。这有助于加速广泛的精度 医学，即。精准癌症诊断、免疫治疗；治疗性基因编辑；新药发现和 验证。该技术可以为对抗传染病提供革命性的进步，包括 HIV/艾滋病和 SARS-CoV-2 介导的 COVID 19。