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

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 与各种癌症的发展和进展以及治疗抗性相关。测序 分析整个细胞外RNA片段组，包括小的非编码RNA（如miRNA）以及 较大的编码和非编码RNA片段（如mRNA、lncRNA和tRNA）可能提供更高的 来源特异性和敏感性分析，这是它们用作癌症生物标志物所必需的。但有 技术问题限制了RNA片段组的完整补体的可视化，其中大部分是 由小RNA（sRNA）和大小小于50个核苷酸的sRNA片段表示， 3 '-磷酸（3'-p）或2 '，3'-环磷酸（2 '，3'-cP）与5 '-羟基（5'-OH）或5 '-磷酸（5'-p）一起沿着 终点。具有这种末端的RNA分子不能被标准测序文库制备物捕获， 被设计为主要与具有5 ′-p/3 ′-OH末端的sRNA（例如，miRNAs），因此， (or隐藏）以避免通过测序检测。为了分析除了miRNA之外的更广谱的sRNA， 对于开发RNA-seq文库制备方案至关重要，该方案能够检测具有以下特征的所有sRNA类别： 它们末端的不同磷酸化状态以及这些sRNA类型之间的区别， 形接头.尽管最近已经描述了一些用于分析RNA片段组的方法，但它们中没有一个是有效的。 提供了允许同时检测所有sRNA类型或单独检测每种类型的选择 使用相同的方法制备文库。此外，这些方法集中于检测特定的sRNA 具有特定末端（例如，5'-OH或3'-cP），并且不能区分磷酸化状态的变化， 相反的sRNA末端。此外，这些方法中的大多数使用需要纯化化合物的反应条件。 在下一个反应步骤之前产生反应产物，这可能导致材料损失和再现性降低。最后， 没有可用于sRNA-seq文库制备的商业试剂盒， RNA片段组学分析。为了解决这些缺点，我们提出了一种先进的方法，称为 RealSeq-RF，代表了我们RealSeq®平台技术的进一步创新发展， miRNA测序文库（https：//www.realseqbiosciences.com/technology）。这一新方法的进展 RealSeq®能够检测所有sRNA类型或其中一种特定类型。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 通过分析和比较来自健康供体血浆的sRNA片段组， 白血病患者因为许多类型的白血病在疾病早期没有明显的症状， 发展微创、早期检测白血病将是其成功的关键。 治疗

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.