Preparation of sequencing libraries for multi-analyte analysis of small RNAs

制备用于小 RNA 多分析物分析的测序文库

• 批准号: 10759916
• 负责人: Sergei A Kazakov
• 金额: $ 30万
• 依托单位: REALSEQ BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-21 至 2024-06-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10759916
• 关键词: Address; Awareness; Bar Codes; Benchmarking; Biochemical Process; Biogenesis; Biological; Biological Markers; Cancer Diagnostics; Cancer Patient; Cells; Circulation; Code; Complement; Data Analyses; Detection; Development; Discrimination; Disease; Evaluation; Genes; Human; Hydroxyl Radical; Length; Libraries; Malignant Neoplasms; Maps; Measures; Messenger RNA; Methods; MicroRNAs; Modeling; Modification; Monitor; Mutation; Nucleotides; Patients; Periodicity; Phase; Phosphorylation; Plasma; Preparation; Process; Protocols documentation; Publishing; RNA; RNA Degradation; RNA Sequences; RNA analysis; RNA-Binding Proteins; RNA-Protein Interaction; RNase protection assay; Randomized; Reaction; Reproducibility; Resistance; Ribonucleases; Sampling; Screening for cancer; Sensitivity and Specificity; Small RNA; Specificity; Survival Rate; Symptoms; Technology; Testing; Tissues; Transfer RNA; United States; Untranslated RNA; Variant; Visualization; Work; cancer biomarkers; cancer therapy; design; detection method; extracellular; improved; innovation; inorganic phosphate; leukemia; miRNA expression profiling; minimally invasive; mortality; novel; novel strategies; technology platform; transcriptome sequencing; tumor

Recent studies show that the mutations and dysregulated biogenesis of coding and non-coding RNA genes are associated with the development and progression as well as therapy resistance for various cancers. Sequencing analysis of the entire extracellular RNA fragmentome including small non-coding RNAs (like miRNA) as well as fragments of larger coding and non-coding RNA (like mRNA, lncRNA and tRNA) could potentially provide higher origin-specificity and sensitivity profiling required for their use as cancer biomarkers. However, there are technical problems limiting a visualization of the full complement of the RNA fragmentome, most of which is represented by small RNAs (sRNA) and sRNA fragments that are less than 50 nucleotides in size and possess 3’-phosphate (3’-p) or 2’,3’-cyclic phosphate (2’,3’-cP) along with 5’-hydroxyl (5’-OH) or 5’-phosphate (5’-p) termini. RNA molecules having such ends cannot be captured by standard sequencing library preparations which were designed to work primarily with sRNAs having 5’-p/3’-OH ends (e.g., miRNAs), and, therefore, are invisible (or hidden) from detection by sequencing. To analyze a broader spectrum of sRNAs in addition to miRNAs, it is essential to develop RNA-seq library preparation protocols that enable detection of all sRNA classes having different phosphorylation states of their ends as well as discrimination between these sRNA types with differing ends. Although some methods for analysis of the RNA fragmentome have been recently described, none of them provided options that would allow detection of either all sRNA types simultaneously or each type separately using the same approach for library preparation. Also, these methods are focused on detecting specific sRNAs with specific ends (e.g., 5’-OH or 3’-cP) and cannot distinguish between variations of phosphorylation status at the opposite sRNA end. Moreover, most of these methods use reaction conditions that require purification of reaction products before next reaction steps that might result in lost materials and reduced reproducibility. Lastly, there are no commercial kits available for sRNA-seq library preparations that would allow comprehensive analysis of RNA fragmentomics. To address these shortcomings, we propose an advanced approach, called RealSeq-RF, that represents further innovative development of our RealSeq® platform technology for making miRNA sequencing libraries (https://www.realseqbiosciences.com/technology). This new approach advances the capability of RealSeq® to allow detection of all sRNA types or one of them specifically. In Phase I we plan to develop the RealSeq-RF approach (initially) using model synthetic sRNAs (ssRNA) and then validate the developed approach by analyzing and comparing the sRNA fragmentome from plasma of healthy donors and leukemia patients. Because many types of leukemia show no obvious symptoms early in the disease, the development of minimally invasive, early-stage detection of leukemia would be a critical for its successful treatment.

最近的研究表明，编码和非编码RNA基因的突变和失调的生物发生是 与各种癌症的发育和进展以及耐药性相关。测序 分析整个细胞外RNA碎片组，包括小非编码RNA（例如miRNA）以及 较大的编码和非编码RNA（例如mRNA，lncRNA和tRNA）的片段可能会提供更高的 起源特异性和敏感性分析需要用作癌症生物标志物。但是，有 技术问题限制了RNA片段完整完成的可视化，其中大多数是 由小RNA（SRNA）和SRNA片段表示，大小小于50核苷酸 3'-磷酸（3'-P）或2'，3'-Cycle磷酸盐（2'，3'-CP）以及5'-羟基（5'-OH）或5'-磷酸（5'-P） 终点站。具有这种末端的RNA分子不能通过标准测序库制剂来捕获 设计用于主要与具有5'-p/3'-OH末端的SRNA（例如miRNA），因此是无形的 （或隐藏）通过测序检测。为了分析除miRNA之外的较广泛的SRNA，它是 制定RNA-seq库制备方案的必不可少的必不可少的必要 其末端的不同磷酸化状态以及与分化的这些SRNA类型之间的歧视 尽管最近已经描述了一些用于分析RNA碎片组的方法，但它们都没有 提供的选项可以单独检测所有SRNA类型或每种类型 使用相同的方法进行图书馆准备。另外，这些方法的重点是检测特定的SRNA 具有特定的末端（例如5'-OH或3'-CP），无法区分磷酸化状态的变化 相反的sRNA端。此外，大多数这些方法都使用需要净化的反应条件 在下一个反应步骤之前的反应产物可能导致材料损失并降低可重复性。最后， SRNA-Seq图书馆准备工作中没有商业套件可以全面 RNA片段学的分析。为了解决这些缺点，我们提出了一种高级方法，称为 Realseq-RF，这代表了我们的Realseq®平台技术的进一步创新开发 mirna测序库（https://www.realseqbiosciences.com/technology）。这种新方法进步 Realseq®允许检测所有SRNA类型或其中一种的能力。在第一阶段，我们计划 使用模型合成SRNA（SSRNA）开发RealSeQ-RF方法（最初），然后验证 通过分析和比较健康捐赠者血浆和 白血病患者。由于许多类型的白血病在疾病初期没有明显的症状，所以 最低侵入性，早期发现白血病的发展对于其成功而言至关重要 治疗。