Small-molecule RNA, including siRNA (small interfering RNA), miRNA (microRNA), piRNA (piwi-interacting RNA), etc., has been awarded "Top Ten Scientific Breakthroughs" and "Top Ten Scientific Progress" by the US "Science" magazine for many times. It is the frontier hotspot of current life science research. A large amount of experimental evidence shows that these small RNAs exist in almost all eukaryotic cells and play an important role in regulating gene expression, cell cycle, and organism development.
In recent years, studies have shown that a variety of small molecule RNAs have 3 'terminal methylation, such as miRNA, siRNA, hc-siRNA (heterochromatic small interfering RNA), ta-siRNA (trans-acting siRNA), and nat-siRNA in plants (natural antisense short interfering RNA), siRNA and piRNA in insects, piRNA in animals. The methylation of the 3 'end of these small molecule RNAs can protect them from a variety of exonuclease, ligase, terminal transferase, polymerase and other enzymes in the cell that can act on the hydroxyl group of the 3' end of the nucleic acid, thereby protecting the small Stability of molecular RNA. Although the 3 'end methylation of these small RNAs does not change the nucleotide sequence, it has brought great challenges to the existing high-throughput detection technology (chip technology, sequencing technology). Because most of the existing commercial high-throughput detection methods are based on enzyme labeling or enzyme ligation, these enzyme reactions need to interact with the 3 'end of small RNA, and the methylation of the 3' end will inhibit the efficiency of the enzyme reaction In the end, the detection results are inaccurate (the chip method is usually a false negative signal, and the sequencing method is usually unable to detect).
Li Jiong's research group of Suzhou Institute of Nanotechnology and Nanobionics, Chinese Academy of Sciences, following the first high-throughput, non-labeled chip method to achieve conventional small-molecule RNA (without modification) published in Nucleic Acids Research in 2011, further confirmed that the method is not subject to small The methylation of the 3 'end of molecular RNA can accurately detect the modified small molecule RNA (as shown in Figures c and d); at the same time, the poly (A) polymerase used in commercial chips is quantified by the 3' end The degree of influence of methylation (Poly (A) polymerase reaction efficiency of methylated small RNA is about 1/24 of conventional small RNA, as shown in Figures a and b).
In addition, this non-labeled chip detection method also inherits and promotes the advantages of detecting unmodified small molecule RNA: 1. Efficiently recognize the single base deletion and redundancy at the end of the small molecule RNA, and the difference of single bases at the 1-3 positions at the end, This is difficult to achieve for conventional chip technology; 2. High sensitivity, detection limit is 20 fM, detection abundance spans 4 orders of magnitude, to meet the detection of most small molecule RNA in organisms; 3. Direct use of total RNA, no pre-required Isolate small-molecule RNA without the need for sample labeling, which greatly reduces the detection time and cost.
The above-mentioned non-labeled chip detection method not only solves the bottleneck problem encountered in the detection of small molecule RNA in existing commercial products, but also provides an ideal solution for high-throughput detection of small molecule RNA methylated at the 3 'end. Promote the functional research of methylated small molecule RNA and contribute to the research and development of "epigenetics" and "post-genome".
The work was recently published in the journal Analytical Chemistry, with strong support from the Chinese Academy of Sciences, the National Fund Committee and the Jiangsu Natural Science Foundation.
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