Preliminary Study On The Labeling Of Prenylated TRNA And Protein

Biological macromolecules are functional molecules that carry all life activities.Biological macromolecules include nucleic acids,proteins,and so forth,which play important roles in the transmission of genetic information and cell biochemical activities.In addition to understanding its basic structure and function,research on varied modifications that covalently attached on biological macromolecules including nucleic acid and proteins has gradually attracted community's attention in recent years.In this dissertation,a special lipid modification with isopentenyl as the backbone has been intensively investigated.On the one hand,we explored the molecular labeling of geranyl(two isopentenyl)t RNAs.A series of geranyl pyrophosphate derivatives were synthesized through chemical synthesis approach,and the"writer"enzyme Sel U was constructed and expressed.After co-incubation with E.coli,we demonstrated that geranyl pyrophosphate substrates(G5 and G6)with azido functionalities could be further tagged by fluorescent dye(DBCO-Cy5)via the copper-free click reaction and confocal fluorescence imaging.Thus,it was proven that geranyl pyrophosphate substrates(G5 and G6)could be successfully recognized by Sel U and covalently linked into t RNA.In addition,another two geranyl pyrophosphates analogues(G2 and G3)are incubated with E.coli.cells.Later,the extracted total t RNA was completely hydrolyzed into nucleosides and analyzed by use of liquid chromatography-mass spectrometry(HPLC-MS)along with comparations with nucleosides G2-s~2U and G3-s~2U.The subsequent qualitative and quantitative analysis of the Sel U-mediated geranylation efficiency with these two analogues have also been tested.On the other hand,we have developed a highly-selective labeling method that directly targets isopentenyl groups using Selectfluor reagent or well-designed 4-phenyl-3H-1,2,4-triazoline-3,5 The(4H)-dione(PTAD)reagent.Via the[2,3]-rearrangement Ene-ligation.After optimization of the reaction condition,we applied it to the selective labeling study of t RNA from E.coli.and bovine serum albumin(BSA).We demonstrated that prenyl-involved[2,3]-rearrangement Ene-ligation using Selectfluor reagent can be used in the direct detection of prenylated t RNA;PTAD and its derivatives can be used to selectively label prenylated nucleic acids or proteins.In short,on the basis of the preliminary research of our research group,we have successfully demonstrated that both the indirect approach,namely"synthesis of geranyl pyrophosphate analog”-“co-incubation”-bioorthogonal labeling experiment",and the direct approach by use of Selectfluor reagent and well-designed and successfully synthesized PTAD reagent.This Ene-ligation relying on prenyl functionality can be utilized for the studies of prenylated nucleic acids and proteins.This work lays a solid foundation for transcriptomics or proteomics research on prenylation modification.The follow-up research is currently underway in the laboratory.