Research On Labeling And Detection Of Prenyl-modified Biomolecules And Functional Regulation Of Nucleic Acids By Reactive Oxygen Species

Currently,over 150 post-transcriptional RNA chemical modifications have been found.Except for methylation in RNA,understanding of the biological functions of other modification types remains in high demand.There is prominent meaning to explore chemical methods for the studies of prenyl groups in nucleic acids or proteins to investigate its biological functions and to elucidate its role in relevant diseases.N⁶-isopentenyladenosine（i⁶A）is one of the hyper-modified adenosines characterized by an isopentenyl chain.Considering the profound impact of i⁶A,methods for precise manipulation will serve as powerful tools for research into the physiological function while avoiding genetic redundancy,and they may also help in the identification of chemical regulators for i⁶A-dependent cellular pathways.Besides,as clinical studies on nucleic acid-based drugs are making rapid progress,small-molecule triggered approaches as valuable complements to photoactivation are worth investigation.The thesis is composed of three parts:Part 1:Development and Application of Prenyl Functionality BasedBiocompatible Ligation.The development of bioorthogonal reaction has greatly facilitated pharmaceutical research such as drug screening,drug synthesis and drug delivery studies.As important metabolic materials in living organisms,prenyl functional groups have prominent compatibility in the physiological environment.In the first part of the paper,we first developed a rapid and biocompatible Alder-Ene ligation based on the natural productβ-caryophyllene containing strain prenyl group and the azo reagent4-phenyl-1,2,4-triazole-3,5-dione（PTAD）.This reaction exhibited excellent solvent compatibility,reaction selectivity and orthogonality,while the secondary reaction kinetic constants was calculated up to 1.46 M^-1 s^-1.Moreover,the reaction can be used orthogonally to the strain-promoted alkyne-azide cycloaddition（SPAAC）for dual protein labeling and in vivo labelling.This naturally occurring strain-promoted prenyl-involved Alder-Ene ligation will be widely used in the fields of chemical and biological research（e.g.antibody-drug conjugate）.Part 2:Development of Chemical Labeling Strategies for Use in the Fluorescence Labeling and Detection of i⁶A.In the second part of dissertation,besides PTAD-based Ene reaction,TMSN₃/Selectfluor-mediated azidofluorination and I₂-induced cyclization were simultaneously developed to selectively chemically modify i⁶A.In i⁶A-containing oligonucleotide and in vitro transcript RNA,fluorescent labelling of i⁶A were successfully performed with the Ene ligation and the azidofluorination cascade SPAAC reaction.Meanwhile,I₂-induced i⁶A mutation sequencing method was initially validated in i⁶A-containing transcript RNA.The establishment of these chemical labelling strategies provides simple and effective tools for conducting i⁶A localization and tracking as well as biological function studies.Part 3:Reactive Oxidative Species-Triggered Regulation of DNA Function.Deoxyribonuclease（DNAzyme）is one of the representative functional nucleic acids,which has been widely used in research for the treatment of viral infectious diseases,tumors and cardiovascular diseases.This part focused on oligonucleotide10-23 DNAzyme,a study on the active regulation of DNAzyme’s base pairing function by reactive oxygen species was proposed.The result showed that the caged group in multiple sites of 10-23 DNAzyme was rapidly removed by the treatment of hydrogen peroxide or N-oxides.The regulation of caged 10-23 DNAzyme T-A base pairing function has been precisely monitored by DNA polymerase chain reaction.This caged strategy will be helpful in study of 10-23 DNAzyme for the treatment of related viral infectious diseases（e.g.,hepatitis B）.In summary,these studies not only open a new avenue to utilize naturally occurring scaffolds as chemical reporters,but also provide new strategies for the localization,labelling and functional regulation of nucleic acids.