Detection Of Deoxyuridine And N1-methyladenosine In Nucleic Acids

Hundreds of modified bases have been found in nucleic acids,which are involved in the functional regulation of organisms together with conventional bases(A,T/U,C,G).There are many functional modifications,such as the methylation of DNA and RNA.In addition,there still are many modified bases whose functions are still in the exploratory stage,such as deoxyuridine(d U)in DNA and N1-methyladenosine(m~1A)in RNA.In this paper,we mainly take d U and m~1A as the research objects to provide a simple and fast detection method for exploring their functions and dynamic changes in organisms.dU in DNA mainly comes from the deamination of cytosine or incorrect incorporation of d UMP.At present,four enzymes have been reported to be involved in the base excision and repair of d U,namely UNG,SMUG1,TDG and MDB4.The presence of d U in the genome is closely related to many life activities and plays an important role in many systems such as antibody maturation,virus life cycle and Drosophila development.Here,we report a method combining UNG enzyme excision for qualitative and quantitative analysis of d U in genomic DNA.First,d U in genomic DNA was selectively transformed into AP site by UNG enzyme,and then labeled with NRNO.After labeling,o-phenylenediamine in NRNO was converted to benzimidazole,and the PET effect disappeared which introducing a strong signal at the far infrared region of 650 nm.Qualitative and quantitative detection of d U could be realized according to the change of fluorescence intensity.Because of steric hindrance effect,it can act as a“roadblock”to abort the primer extension to provide a detailed information about the d U site.In addition,the probe can also be used for fluorescence imaging analysis of d U concentration changes by two-photon confocal fluorescence microscopy.In RNA,m~1A is the second reversible modification found in addition to m~6A.The modification of m~1A in t RNA plays an important role in the structure and stability of t RNA.m~1A in 5'cap and 5'UTR of m RNA may play an important role in promoting translation.In consideration of its crucial roles,we developed a detection system based on CRISPR-Cas13a for m~1A detection in RNA.When m~1A is present in the target RNA sequence,it will affect its complementary pairing with cr RNA,thus it cannot activate the RNase activity of Cas-13a protein to cleave a short quenched-fluorescent reporter RNA to release fluorescent signals.When the site is normal A,it can activate the activity of RNase and release fluorescent signals.Therefore,we can quantitatively andqualitatively analyze the m~1A in RNA by fluorescence signal intensity analysis.In addition,with the assistance of demethylase Alk B,we can identify m~1A at the position of 1309 in 28s r RNA.Finally,it can also be used to study the dynamic demethylation process of m~1A by adjusting the demethylation time of Alk B.