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

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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.