Synthesis And Detection Of 5-formyluracil In DNA

Natural pyrimidine modifications play a key role in the regulation of gene expression in eukaryotes and prokaryotes,and their dysregulation may lead to disease.With the continuous use of chemical tools,new detection technologies have greatly promoted the research progress of natural pyrimidine modifications.In addition to 5-methylcytosine,5-hydroxymethylcytosine,5-formylcytosine,and 5-carboxycytosine,the organism also contains a small but extremely important 5-hydroxyuracil and 5-formyluracil.5-Formyluracil is found in bacteriophages,prokaryotes,and mammalian cells.And content of 5-formyluracil is higher in certain cancer tissues than in adjacent tissues.The content of 5-formyluracil in different cell tissues may have cell type specificity.The level of natural pyrimidine modifications in blood DNA can be studied as a marker for breast cancer.Natural pyrimidine modifications are not only intermediates for DNA demethylation or oxidative damage products,but also act as modulators of gene expression.They can be produced from products of active oxidation of enzymes in living organisms,and can also be passively caused by damage of nucleic acids by ultraviolet rays,gamma rays,reactive oxygen radicals,etc.in the environment.Therefore,the development of more effective chemical tools will help us better understand the dynamic changes of natural pyrimidine modifications in vivo.We designed and synthesized a new 5-formyluracil phosphoramidite monomer.This new phosphoramidite monomer was used for synthesis of 5-formyluracil oligonucleotides via traditional DNA solid phase synthesis techniques.Through matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high resolution mass spectrometry data of enzymatically degraded oligonucleotides,we verified that we did indeed synthesize oligonucleotides containing 5-formyluracil.The method has the advantages of simplicity,high efficiency,non-toxicity,easy operation,and easy availability of starting materials.In addition,sodium iodine oxidant is avoided in the subsequent purification of nucleic acid,which enhances the synthesis efficiency of the oligonucleotide chain and reduces the difficulty of separation.And we created a high selectivity,efficient,colorimetric,qualitatively and quantitatively detection methods for 5-formyluracil in single-and double-stranded ODNs.Moreover,we also applied this method to the content detection of complex real samples after gamma ray irradiation.Combining Potassium pertechnetate which can selectively oxidize 5-hydroxymethyluracil to 5-formyluracil,we also designed an oxidation-labeling protocol and extended this method to the study of 5-hydroxymethyluracil in nucleic acids.Finally,we designed and synthesized a new biotinylated naphthalimide-o-phenylenediamine molecule.Due to the photo-induced electron transfer(PET)effect,excess electrons in o-phenylenediamine can quench the fluorescence of naphthalimide.And o-phenylenediamine can also react with 5-formyluracil very selectively and efficiently.Therefore,in our design,o-phenylenediamine can not only quench the naphthalimide fluorescence,but o-phenylenediamine directly attached to the naphthalimide can be very effective and selective towards 5-formyluracil.This molecule not only can fluorescently label 5-formyluracil under physiological conditions,but also can enrich DNA fragments containing 5-formyluracil in nucleic acid samples.It can also be used for cell imaging after exposure to gamma irradiation and getting quantitative information for specific sites of 5-formyluracil by polymerase primer extension experiments.In addition,the ideas for 5-formyluracil from synthesis to detection are expected to be applicable to other natural base modifications.