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

> **NIH NIH R43** · REALSEQ BIOSCIENCES, INC. · 2023 · $300,000

## Abstract

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 crit...

## Key facts

- **NIH application ID:** 10759916
- **Project number:** 1R43HG013284-01
- **Recipient organization:** REALSEQ BIOSCIENCES, INC.
- **Principal Investigator:** Sergei A Kazakov
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $300,000
- **Award type:** 1
- **Project period:** 2023-09-21 → 2024-06-20

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10759916

## Citation

> US National Institutes of Health, RePORTER application 10759916, Preparation of sequencing libraries for multi-analyte analysis of small RNAs (1R43HG013284-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10759916. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*