Exploring The Distance Tolerance Of Bivalent Probes On The Cell Surface By DNA-based Nanoruler

Cell-surface receptors,also known as transmembrane receptors,are proteins that attach to cell membranes.Cell surface receptors can bind to external ligand molecules(such as hormones,neurotransmitters,cytokines,growth factors,etc.)and transform extracellular signals into intracellular signals,causing changes in cell metabolism and activity.Current studies have demonstrated that many cell membrane receptors are activated by ligand-induced dimerization or oligomerization,which controls cell growth,differentiation,and movement,and their dysregulation is associated with many human diseases,including various cancers.Therefore,the exploration of artificial regulation of cell membrane receptor dimerization is of great significance for the development of disease treatment.Among the strategies reported so far,considerable progress has been made in the regulation and activation of receptors by constructing bivalent probes using aptamers.The Bivalent aptamer probe is a structure formed by the assembly of two units with the same or different aptamer motifs by connecting molecules.Due to the programmability of nucleic acid molecules,it is easy to modify and assemble the bivalent probe by using nucleic acid connecting molecules with different properties.Although bivalent aptamer probes have achieved varying degrees of success in inducing receptor protein aggregation and improving binding affinity to the target,little is known about how the length,chemical properties,and flexibility of the link chain that connects the two aptamers in the probe structure affect the interaction between the bivalent probes and the protein.Here,we assembled bivalent aptamer probes with different structures to investigate the effects of the length,and rigidity of the connection structure on the affinity of the bivalent probes,and to explore the distance limit the bivalent probes on the cell surface receptors.The research has certain guiding significance to design the bivalent probe,bivalent aptamer drugs,cell surface receptor interaction and receptor density.The specific work contents are as follows:Chapter 2: The effects of different mutation sites on the affinity of aptamer were investigated.The aptamer is a special nucleic acid targeting proteins specified by forming a special tertiary structure.Aptamer is a very promising molecular recognition tool has been widely used in the fields of biological analysis,biological imaging,drug delivery,disease diagnosis,and treatment.In this chapter,Sgc-8 and XQ-2d were selected with changing the affinity between aptamers and CCRF-CEM cells by mutating the special sites on their sequences.The results showed that when the mutated base site was the key region of aptamer binding to the target protein,or the change of sequence affected the conformational stability of aptamer,the affinity of aptamer would is weakened to varying degrees,and even could not be targeted to cells.In this chapter,the key region of aptamer binding was found through an affinity experiment,which provided a guarantee for the reasonable construction of the bivalent probe in the next step.Chapter 3: Exploring the distance tolerance of bivalent aptamer probes assembled by aptamers with different affinity on the cell surface.Based on this work in Chapter 2,two aptamers were selected and named Sgc8-m1 and Sgc8-m2 respectively in this chapter.The affinity of these two aptamers is weaker than Sgc-8 in turn.The three aptamers were assembled into bivalent aptamer probes with distances of 20 bp,30 bp,40 bp,50 bp,60 bp,80 bp,90 bp,and 100 bp,respectively.The distance tolerance of the bivalent probes on the cell surface was investigated by competitive experiments when the affinity of monovalent aptamers was different.The results showed that the limit distance of the bivalent probes on the cell surface was positively correlated with the affinity of the monovalent aptamers,and the bivalent effect disappeared when the distance exceeded a certain distance.This work in this chapter can provide important guidance for the design of bivalent probes,the development of bivalent aptamer drugs,and the study of cell surface receptor interactions.