| Chemical modifications on biological macromolecules play important roles in precise fine-tuning of their structures and functions.Studies have shown that the chemical modifications on nucleic acids often have a very significant regulatory effect on the biological functions of the relevant genes.The study of the spatiotemporal and reversible dynamic chemical modification process in nucleic acids is important for understanding life processes,especially the relationship between the distribution and amount of nucleic acid modifications and human diseases has crucial significance.Sulfur modification is a type of chemical modification that has been found to be existed extensively in living organisms.Studies have shown that sulfur-modified nucleic acid plays a role in stabilizing nucleic acid structure and improving the accuracy of translation in organisms.However,there are very few reports on the sulfur modification of nucleic acids,which are mainly restricted by limited research tools.For example,as for nucleic acids containing s~4U modification,Duffy et al reported an efficient and highly-selective enrichment method based on sulfur atom exchange strategy.In order to comprehensively and accurately clarify the biological functions of sulfur-modified nucleic acids in vivo and their association with human diseases,it is of great research significance to study specific labeling and enrichment methods.s~2T and m~5s~2U are two typical sulfur-modifications found in nucleic acids.The characteristic is that the modification is located at position 2 of the pyrimidine base,which is different from the nucleoside s~4U modified by the sulfur atom at position 4.By reviewing literature,we notice that there is no report on a highly selective and efficient labeling method for modified nucleic acids containing s~2T and m~5s~2U.Therefore,this dissertation mainly focuses on screening of highly selective and efficient labeling methods containing s~2T or m~5s~2U nucleic acids.In addition,it is reported that prenyl-modification are widely present in natural products,nucleic acids and proteins.Our research group has recently developed an Ene-ligation forprenylated molecules.This dissertation also preliminary illustrated the application of this Ene-ligation to the fluorescent labeling of citronellol-modified proteins.Through the screening of a series of alkylating reagents,we found that the reaction efficiency of iodoacetyl reagent was the best candidate.After exploring the conditions,we found that the iodoacetyl reagent can rapidly undergo alkylation reaction with m~5s~2U-modified nucleic acid in a neutral aqueous solution within five minutes at 95~oC.Based on this result,we later use fluorescein FITC-derived iodoacetamide reagent(2.33)to successfully label s~2T-or m~5s~2U-modified nucleic acids.This is the first example of labeling of nucleic acids containing s~2T or m~5s~2U modifications.This method has the characteristics of good selectivity and relatively high efficiency,and lays a solid foundation for the subsequent transcriptomics research of sulfur atom-modified nucleic acids.In addition,based on the Ene-ligation developed by our research group,this dissertation explored the citronellol pre-targeted BSA protein,and then applied the PTAD-DBCO-Cy5 reagent synthesized by our group to realize the labeling of the citronellol-modified protein. |
