A kind of RNA 4-mercaptouracil specific labeling method and its application
A technology of mercaptouracil and RNA4-, which is applied in the field of nucleic acid chemistry, can solve the problems of low reaction efficiency, harsh reaction conditions, complicated process, etc., and achieve the effect of good selectivity and strong specificity
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Embodiment 1
[0069] Example 1: Using the addition and elimination reaction of RNA 4sU using hydrazine hydrate according to the present invention, a structure similar to cytosine was obtained.
[0070] In vitro transcription of RNA with a 4sU site, the 4 S U-RNA was reacted with hydrazine hydrate, and the reaction conditions were: water as solvent, 10mM DTT buffer, and heating at 50°C for 1.5h. After the reaction, the RNA was recovered by alcohol precipitation. The labeled RNA was reacted with rSAP and RNAse I at 37 °C for 12 h, and the nucleic acid was degraded into nucleosides. Finally, various nucleosides in the samples were detected by LC-MS / MS.
[0071] see figure 2 , is 4 S UV absorption spectra before and after the reaction of U with hydrazine hydrate, it can be seen from this picture that hydrazine hydrate can convert 4 S U was completely reacted, and the new product cytidine analog C* was obtained.
[0072] HPLC mass spectrogram of nucleosides after RNA degradation see ima...
Embodiment 2
[0074] Example 2: Single-base recognition of 4sU by the addition and elimination reaction of 4sU using hydrazine hydrate according to the present invention
[0075] The RNA with 4sU site was transcribed in vitro, and the 4Su-RNA was reacted with hydrazine hydrate. The reaction conditions were water as solvent, 10mM DTTbuffer, and heating at 50°C for 1.5h. After the reaction, the RNA was recovered by alcohol precipitation. The labeled RNA was reverse transcribed and PCR was performed. PCR products were characterized by 15% PAGE and sequencing. A mutation from T to C occurred at the position of the original 4sU. Therefore, this method realizes single-base recognition of 4sU.
Embodiment 3
[0076] Example 3: Identification of modification positions of naturally occurring 4-mercaptouracil
[0077] Total RNA was extracted from Escherichia coli, and total RNA was reacted with hydrazine hydrate. The reaction conditions were water as solvent, 10 mM DTT buffer was added, and the reaction was carried out at 50 °C for 1.5 h. Use specific primers of known sequence to tRNA val Reverse transcription, PCR, and next-generation sequencing were performed to identify and confirm the location of the naturally occurring modified 4-mercaptouridine.
[0078] For the process of subcloning in this example, see Figure 10 , for next-generation sequencing results of subcloned plaques, see Figure 11 , which shows the naturally occurring 4 S The U site changed from the original A:T pairing to the C:T pairing, indicating that the method of the present invention can recognize the naturally occurring 4 S U site.
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