Nucleic acid modification refers to the modification of DNA or RNA bases by introducing additional chemical groups. It does not change the arrangement of the gene sequence but greatly improves the ability of the bases to encode genetic information. In recent years, the role of nucleic acid modifications in the regulation of gene expression has attracted increasing attention. The modified bases serve as markers on DNA or RNA and are recognized by specific proteins, participating in the regulation of gene expression and further affecting physiological functions. However, base modification usually does not change the base pairing pattern, so it is difficult to be directly identified by molecular biology methods such as PCR. For many years, a lack of methodologies has seriously hindered the development of nucleic acid modification research. Recently, with the continuous advancement of new technologies, there has been a breakthrough in the problem of accurate identification and localization of various modifications on DNA and RNA. At the same time, inspired by gene editing tools, researchers have developed a series of new nucleic acid modification editing tools by integrating gene targeting tools and modification-related effector proteins, trying to change the modification status of target genes to study nucleic acid modification mechanisms at specific sites.
Each of the four bases of DNA has different modifications, with the 5-methylcytosine (5mC) modification being the most abundant. As an epigenetic mark, 5mC is closely linked to other chromatin factors, involved in a variety of developmental and physiological processes, and even exerts extensive effects on gene expression in human diseases. In mammals, the enzymes responsible for the addition and removal of 5mC modifications have been clearly reported. Among them, DNMT1/3A/3B proteins are methyltransferases and are the "writers" of 5mC, while TET1/2/3 proteins are demethylases and are the "eraser" of 5mC. These effector proteins, known as "writers" and "erasers," provide effective tools for manipulating the modification state of DNA and may become potential therapeutic targets. Other modifications such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), etc., although less abundant, are also considered to have important regulation.
N6-methyl modification (6mA) is another important DNA modification. It was considered to be the most common DNA modification in bacteria. However, recent studies have found that this modification also exists in some eukaryotes. The proportion of modifications is much smaller than in bacteria. In bacteria, most DNA methyltransferases belong to the restriction-modification (RM) system. The classic function of this system is to defend against the invasion of foreign nucleic acids. For example, after virus invasion, the nucleic acid will be released into the cell. The restriction-modification (RM) system recognizes viral nucleic acids and cleaves them. In addition, there are some "individual" DNA methyltransferases that may have originated from the ancestral restriction-modification (RM) system, but they lost their restriction endonuclease partners during evolution. One typical example is Dam methyltransferase, which methylates adenosine to 6mA on the 5-GATC-3' motif and is equivalent to the writer protein in bacteria. In addition to Dam, there is also a large class of DNA methyltransferases that act on specific sequence motifs, and the corresponding demethylases do not exist in bacteria.
Among approximately 150 RNA modifications, m6A is the most abundant modification on mRNA and plays an important role in the regulation of gene expression. Although the existence of m6A in eukaryotes has long been identified, its specific distribution has been unclear due to the lack of accurate localization methods. It was not until recent breakthroughs in new identification technology that the distribution of m6A in mRNA was determined. With the development of m6A-specific antibodies, researchers began to study its distribution and related effector proteins from a genome-wide perspective. To date, the key regulators involved in the m6A pathway are well studied and elucidated in mammals, including METTL3/14 methylase and related proteins that act as "writer" proteins to add methyl modifications at desired positions. FTO/ALKBH5 demethylase acts as an "eraser" to delete modifications, and YTH family proteins and related proteins act as "reader" proteins to decode.
Fig.1 Methylation and demethylation of m6A in RNA (Fu Y., He C. 2012).
Methods for Positioning Nucleic Acid Modification
With the deepening of life science research, the role of nucleic acid modification in the regulation of gene expression has attracted more and more attention. Different from the regulation of coding nucleic acids, it has a special and huge regulatory mechanism, and analyzing the genomic location of nucleotide modifications is the key to studying its functions. Since there are many types of nucleic acid modifications and their corresponding chemical properties are different, the detection methods for each modification are also very different. Taking m6A as an example, it has basically the same composition as the unmodified A base, and it cannot be distinguished from the unmodified A base through ordinary base pairing strategies. Therefore, many new methods have emerged in recent years to map various modifications of the genome or transcriptome. These methods are usually classified into three categories, namely antibody enrichment method, chemical reaction method, and enzyme recognition reaction method.
The principle of the antibody enrichment method is to use antibodies with relatively high specificity to have a high affinity for specifically modified DNA or RNA fragments, which facilitates subsequent co-immunoprecipitation enrichment to obtain the signal of the modified fragments. After immunoprecipitation, the enriched fragment has more modifications than the background sequence. Through high-throughput sequencing and bioinformatics analysis of the enriched fragment and the background fragment, the number of modification sites can be estimated. These enrichment methods have been widely used in the analysis of various modifications, including 5mC in DNA (MeDIP-seq) and m6A (MeRlP-seq) in RNA, etc. However, the biggest limitation of the antibody enrichment-based method is the low resolution, which can only map to ~100 nt length range but cannot map to the accurate position.
Chemical reaction methods are distinguished based on the characteristics of modified bases or unmodified bases themselves participating in chemical reactions. For example, the most successful and widely used chemical reaction method in modification detection is 5mC bisulfite sequencing (BS-seq). Its working principle is that after treatment with the chemical reagent bisulfite, cytosine (C) is converted into uracil (U), after reverse transcription and PCR amplification and conversion, will be recognized as thymine (T), but it will not affect 5mC. Through this method, the distribution map of 5mC in the genome can be analyzed.
The enzyme recognition reaction method uses the characteristics of natural enzymes to recognize certain types of modifications in the body, so as to perform cleavage or stop the cleavage reaction to distinguish between modified bases and unmodified bases. For example, restriction endonucleases belong to the restriction modification system (RM system), which uses modifications as markers to identify endogenous DNA and foreign invaders. Taking advantage of this property, researchers have established methods to map 5mC and 6mA based on certain modification-sensitive restriction enzymes.
References
- Qi C.; et al. Analytical methods for locating modifications in nucleic acids. Chinese Chemical Letters. 2019, 30(9): 1618-1626.
- Chen K.; et al. Nucleic acid modifications in regulation of gene expression. Cell Chemical Biology. 2016, 23(1): 74-85.
- Fu Y.; He C. Nucleic acid modifications with epigenetic significance. Current Opinion In Chemical Biology. 2012, 16(5-6): 516-524.
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