MicroRNA (miRNA) is a class of small (approximately 22 nt) non-coding RNA. Its short sequence contains rich information, which involves almost all aspects of life processes, including cell growth, development, metabolism, viral infection, immune response, and tumor initiation and metastasis (Kim et al., 2018). By binding to their complementary mRNA target, miRNA regulates gene expression to achieve these functions. In addition to the sequence combination of four canonical bases including A, U, C, and G, the base modification existing on miRNA also plays an important role in this process. Recent studies reveal that m6A modification in miRNA can affect the process of miRNA biogenesis (Alarcon et al., 2015), and its aberrant level of existence are related to gastrointestinal cancers (Konno et al., 2019). Therefore, deciphering the sequence and modification of miRNA by directly sequencing a single miRNA molecule, can provide valuable information for understanding the life process and cancer diagnosis.
Due to the short length of miRNA, the prior art fails to read miRNA sequences directly at the single-molecule level. Conventional method for miRNA sequencing requires reverse transcription followed with amplification. This indirect sequencing method will cause the loss of modification information. Direct sequencing of miRNA is still a challenge to researchers, which is not conducive to precisely locate modifications in miRNAs by sequencing technique. Moreover, multiple chemical modifications on a single miRNA molecule cannot be sequenced simultaneously in principle. Nanopore sequencing, which emerges as a single-molecule sequencing technology, can read nucleic acid sequence based on their physical and chemical properties without the need for amplification. It is the most direct sequencing method for nucleic acids. However, the current nanopore sequencing technology concentrate more on the direct sequencing of long read length of genomes or transcriptomes. There is no related report on the direct sequencing of very short non-coding RNA. At the technical level, current nanopore sequencing platforms are also not suitable for analyzing very short non-coding RNAs such as miRNA.
Recently, Shuo Huang group achieved the first single-molecule sequencing of miRNA using Nanopore Induced Phase-shift Sequencing (NIPSS). The sequence and modification information for miRNA can be acquired simultaneously by this method (Figure 1). NIPSS is a new versatile sequencing method developed by Prof. Shuo Huang's group in recent year (Yan et al., 2019). Unlike existing nanopore sequencing technology, which can only sequence genomes or transcriptomes, NIPSS can be used for directly sequencing any single-molecule analyte that passes through the nanopore. Although the read length for current NIPSS is only 15 nt, it is very close to the full length of mature miRNA (22 nt), which shows technical feasibility for NIPSS sequencing. This concept is verified successfully in the study.
Figure 1. A schematic diagram of NIPSS strategy for direct miRNA sequencing and the corresponding signal.
In this work, the chimeric DNA-miRNA was used as template strand for nanopore sequencing (Figure 2). The chimeric strand can be constructed by ligating the 3’ end of miRNA to the 5’ end of DNA using T4 RNA ligase. During the process of sequencing, the DNA polymerase synthesis is initiated, which enzymatically drives the chimeric strand to the cis side. Utilizing the phase shift, the tethered miRNA passes through the nanopore constriction in steps equivalent to a single nucleotide and miRNA sequence is directly read by nanopore. In principle, the read length of the NIPSS is determined by the phase shift between the polymerase synthesis site and the nanopore constriction. This read length is equivalent to the height of the MspA nanopore used in this work, and it also corresponds to 15 bases at the 3’ end of miRNA. Although it fails to cover the full length of miRNA, the read length is sufficient to directly distinguish 99.5% of existing human miRNAs according to results from bioinformatics analysis (http://www.mirbase.org/). As a proof of concept, the research team chose to sequence several tumor-related miRNAs, including the oncomiR miR-21 and its isomiR miR-21 + U (3’ mono-uridylation product of miR-21), and tumor suppressor let-7a. All tested miRNAs gave characteristic sequencing signals corresponding to their respective sequences. In practice, NIPSS clearly discriminates between miR-21, let-7a and miR-21+U. As the first single-molecule miRNA sequencing in the field, the high resolution is shown in NIPSS, which may inspire other applications, such as discriminating miRNA family members or single-base nucleotide variation.
Figure 2. Construction of DNA-miRNA template using enzymatic ligation method. The obvious band marked as DNA-miRNA in the gel represents the constructed chimeric strand.
The research team furtherly explored the feasibility of NIPSS for detecting modifications existing on miRNA sequences. M6A is a widely watched epigenetic modification of RNA and is routinely found in mRNA. Recent studies reveal that m6A also exist in mature miRNAs and have biological functions. However, it is challenging for current method to locate m6A in miRNA. In this work, the research team introduced a single m6A modified base (Figure 3) into the miR-21 strand as a proof of concept, and observed specific sequencing signals generated by m6A in the further NIPSS experiment. M6A shows higher signal characteristics than the canonical adenosine, which can be precisely located in the miRNA sequence. Compared with the traditional next generation sequencing, NIPSS can get information of miRNA sequences at the single-molecule level, and retains natural modifications which are directly detected by the nanopore. It is currently the only method for detecting miRNA modification using direct single-molecule sequencing.
Figure 3. Detecting m6A modification within miRNA.
In addition, Prof. Shuo Huang's team has been working to develop low-cost, high-throughput detection devices based on nanopore imaging technique in recent years (Wang et al., 2019). It is expected to be combined with NIPSS method in the future to achieve single molecule miRNA sequencing with high-throughput.
The work was titled Direct microRNA sequencing using Nanopore Induced Phase-shift Sequencing (NIPSS) , and a related paper was published in Cell Press's interdisciplinary journal iScience(DOI: https://doi.org/10.1016/j.isci.2020.100916). Jinyue Zhang and Shuanghong Yan, phD students in the School of Chemistry and Chemical Engineering, are the co-first author of the paper, and Prof. Shuo Huang is the corresponding author. This work was funded by National Natural Science Foundation of China (Grant No. 91753108, No.21675083, No. 31972917), Fundamental Research Funds for the Central Universities (Grant No. 020514380142, No. 020514380174), State Key Laboratory of Analytical Chemistry for Life Science (Grant No. 5431ZZXM1804, No. 5431ZZXM1902), Excellent Research Program of Nanjing University (Grant No. ZYJH004), 1000 Plan Youth Talent Program of China, Programs for high-level entrepreneurial and innovative talents introduction of Jiangsu Province. Technology innovation fund program of Nanjing University.