Construction Of MicroRNA Probes With 3'-Chemcial Modifications And Investigation Of Their Intracellular Function

microRNAs(miRNAs)are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level.miRNAs recognize their target mRNAs through sequence complementarity and function by inducing translational repression or degradation of target mRNAs.Since miRNAs are extensively involved in regulation of gene expression,they are closely related to both normal physiologies and pathologies.Biomedical investigations on miRNAs are thus of great interest to researchers in recent years.However,the effect of chemical modifications on miRNA function or behavior still remains elusive.Here,to address this issue,we synthesized miRNAs with different chemical modifications at the 3'-end and explored their intracellular function and behavior in details.1.miRNAs modified with small molecule at 3'-end and their intracellular function.In order to probe miRNA pathway,miRNA probes with different tags are usually prepared and used.miRNAs with 3'-tag is the most often used miRNA probes,whereas the effect of 3'-tag on miRNA function is still unknown.Therefore,we first modified miRNAs with different fluorophores and biotinylated tags at the 3'-end.To investigate their biological function,we developed cellular reporter systems by using luciferase reporter genes for miRNAs,by which we measured and compared the function of these modified miRNAs.2.miRNAs modified with cell penetrating peptides at 3'-end and their intracellular behaviours.Synthetic miRNA mimics that can replace endogenous miRNAs have been widely used to regulate miRNAs and achieve therapeutic outcomes.However,the negative charge of synthetic miRNAs prevents them from crossing cellular membrane.To address this issue,additional carriers such as lipids and nanovectors are often used.However,miRNAs are usually trapped in lysosome when using exogenous carriers to transport them into cells,leading to degradation of delivered miRNAs.Cell-penetrating peptides(CPPs)are short peptides that facilitate cellular uptake of various biomolecules.Here,we synthesized several CPPs and attached them to the 3'-end of miRNA through photo-click chemistry.The intracellular behavior and function of these modified miRNAs were then investigated through fluorescence tracking and measurement of their biological function with cellular reporter systems.