Interaction Analysis Of PUF Protein And Its Target By FRET With Engineered ECFP And FAM-Labeled Oligonucleotides

Within the cell,the interaction between various biomolecules is very important for regulating and maintaining the normal growth,development and reproduction of the cell.F?rster or fluorescence resonance energy transfer(FRET)is a physical phenomenon that requires non-radiative energy transfer between a pair of molecules,which depends on the distance and the emission of photons from the donor to the acceptor fluorophore and reabsorbance.The efficiency of the energy transfer is highly dependent on the choice of donor and acceptor fluorophores.Fluorescent proteins are one of the most popular fluorophores and can form fusion proteins with different protein molecules.Organic fluorophores can be used to label target nucleic acids.FRET technology can be used to analyze the interaction between proteins,as well as the interaction between proteins and other molecules.The PUF protein family is a highly conserved protein that belongs to a broad family of eukaryotic RNA binding proteins.They regulate the life cycle of target m RNA by binding to the 3'-untranslated region(3'-UTR)of the target m RNA and regulating its degradation.Different PUF proteins have specific binding sites called RNA binding scaffolds and PUF functional domains,which can regulate the expression of target RNA.In the process of long-term evolution,PUF protein binding specificity,regulation and target have diversified in eukaryotes.Scientists have analyzed the corresponding relationship between the amino acid sequence of the PUF protein and the recognition nucleotide sequence,laying the foundation for the artificial design of PUF protein and its application in identifying and binding specific RNA sequences.In this study,we first designed a new cyan fluorescent protein mutCFP,by site-directed mutagenesis at positions 64,65,66,and 72 of the green fluorescent protein sequences,and analyzed its emission spectrum.This mutCFP produces a single-peak emission spectrum at490nm,which is different from the double-peak emission spectrum of cyan fluorescent protein at 476nm and 500nm.mutCFP can be used for FRET as a fluorescent donor of FAM fluorophore.We constructed PUF3-mutCFP fusion protein expression vector,purified the fusion protein,and analyze its interaction with the target oligonucleotides.PUF3 protein can bind12 oligonucleotides(GATCGGCAGCGA).We fused the mutCFP coding sequence at the 3'-end of PUF3 coding sequence and expressed this fusion protein in E.coli.The fusion protein PUF3-mutCFP was obtained after separation and purification.We designed and synthesized oligonucleotides with different lengths of adenylates between the 6-FAM label and the PUF recognition site,and used the mutCFP in the fusion protein PUF3-mutCFP as the fluorescence donor,and 6-FAM as the fluorescence receptor for FRET analysis.As a result,we found that PUF3 has highest binding affinity(K_d=163.3 nM)with a linker of 9adenlylates between FAM and the target sequence compared with the 15,20 and 25adenylates;it has highest binding affinity(K_d=44.3 nM)for 12 oligonucleotides target compared with the 6,8 and 10 oligonucleotides.The effect of pH,glycerol and reducing agents on the interaction between PUF3 and target oligonucleotides was further studied using FRET.Through the analysis of dissociation constant measurement,it was found that the affinity of PUF3 and the target oligonucleotides is affected by these factors,and it has a stronger affinity under the conditions of pH8,20%glycerol and 2 m M DTT.In this study,FRET technology was used to analyze the interaction between PUF3 and the target oligonucleotides,such as the effect of the length of the nucleotide chain between the 6-FAM label and the PUF recognition site,and to evaluate different pH,glycerol and reducing agents.And the potential impacts on its interaction were also evaluated.In summary,this study established an interaction analysis system for the interaction of RNA binding proteins and target nucleic acids,which is a foundation for the in vitro analysis of the interactions between the biological macromolecules and the in-depth exploration of their interaction functions in vivo.