Peking University

Bo Xia

Chengqi Yi

Chenxu Zhu

Ankun Zhou

Disclosed are a method for marking 5-formyl cytosine and the use thereof in single base resolution sequencing. The method for marking the 5-formyl cytosine comprises the following steps of: (1) preparing a DNA or RNA sample; and (2) mixing the DNA or RNA sample with a buffer solution and a compound R&#x2014;CH&#x2014;CN to obtain a marking reaction system; and reacting the compound R&#x2014;CH&#x2014;CN therein with the 5-formyl cytosine in DNA and RNA molecules, and thereby achieving the marking of the 5-formyl cytosine; the reaction process is as in (I) below: wherein, Ris an electron withdrawing group next to the CHgroup, preferably &#x2014;CN, (II) or (III), and more preferably &#x2014;CN; R is a DNA or RNA molecule connected to the 5-formyl cytosine; and the pH value of the marking reaction system is 7.5-9. On this basis, also provided in the present invention is a sequencing analysis method for the 5-formyl cytosine. The method can be implemented at a single cell level, and can achieve the sequencing of single-base resolution levels.

Method for marking 5-formyl cytosine and use thereof in single base resolution sequencing

The present invention relates to a 5-formylcytosine specific chemical labeling method and related applications in aspects such as sequencing, detection, imaging, and diagnosis. In the method, a condensation reaction occurs between an active methylene group in an active methylene compound containing a side-chain reactive group and an aldehyde group in 5-formylcytosine or a 1-substituted derivative of 5-formylcytosine, and at the same time an intramolecular reaction occurs between the side-chain reactive group of the active methylene compound and a 4-amino group of cytosine to implement ring closing. By means of the 5-formylcytosine specific chemical labeling method and related compounds of the present invention, detection of the content of 5-formylcytosine in nucleic acid molecules, and specific concentration of 5-formylcytosine-containing nucleic acid samples, and analysis of sequence distribution information of 5-formylcytosine and/or single-base resolution sequence information in nucleic acid molecules and the like may be implemented. The present invention provides various effective research methods in the research fields of epigenetics and nucleic acid biochemistry.

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Beijing, China

Bo Xia