Using Recombined RNA Aptamers to Specifically Change O‐GlcNAcylation on a Protein

Abstract

O‐linked β‐D‐N‐acetylglucosamine (O‐GlcNAc) is an abundant post‐translational modification (PTM) that has been reported on more than 4,000 intracellular proteins. In contrast, there are only two enzymes known to carry out its addition and removal: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). And it remains poorly understood how these enzymes recognize each of their substrates. As a result, researchers are limited in experimental approaches that specifically change O‐GlcNAc levels on a protein, and this is greatly hindering us from dissecting the regulatory roles of O‐GlcNAc. Once called “nucleic acid antibodies”, aptamers are usually short single‐stranded DNA or RNA, which bind their targets with high affinities and specificities. In this research, we developed a method that is capable of changing O‐GlcNAcylation on a specific protein, using recombined RNA aptamers. We performed Systematic Evolution of Ligands by Exponential Enrichment (SELEX), an iterative in vitro selection with a random RNA library, and identified an RNA sequence that recognizes the nucleocytoplasmic isoform of OGT (ncOGT) with high affinity (Kd~50nM). This aptamer binds to the tetratricopeptide repeat (TPR) domain of ncOGT and does not impair its enzymatic activity. We recombined this aptamer with another aptamer that recognizes GFP, and overexpressed the recombined aptamer together with a GFP‐tagged protein in HEK293T cells. Data from Immunoprecipitation (IP) and Western Blot showed that O‐GlcNAcylation on the GFP‐tagged protein was significantly increased in the presence of this recombined aptamer. The interaction between OGT and the GFP‐tagged protein was promoted as well, based on Co‐Immunoprecipitation (Co‐IP) results. Using this method, we were able to specifically increase O‐GlcNAcylation on several GFP‐tagged proteins, including transcription factors and kinases. Taken together, these results suggest recombined RNA aptamers as promising tools to specifically change O‐GlcNAcylation on individual proteins, which will further provide researchers a method to study the regulatory function of O‐GlcNAc with more details.Support or Funding InformationSupported by NIH P01HL107153, R01DK61671 and R01GM116891. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.