Enzymatic Synthesis of Reactive RNA Probes Containing Squaramate-Linked Cytidine for Bioconjugations and Cross-Linking with Lysine-Containing Peptides and Proteins

Abstract We designed and synthesized cytidine 5′-O-triphosphate bearing a reactive squaramate group attached to position 5 of pyrimidine (CESQTP) and used it as substrate for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using engineered TGK DNA polymerase were used for synthesis of RNA probes bearing one to eight reactive modifications. The squaramate moiety speficially reacts with primary amines to form stable amide bond. Thus, the reactive RNA probes underwent bioconjugations with amine-linked fluorophore or with a lysine-containing peptide. Cross-linking reactions of squaramate-modified RNA probes with RNA-binding proteins including several viral RNA polymerases or HIV reverse transcriptase proceeded with moderate to good conversions. Inhibition of RNA-depending RNA polymerases from Japanese Encephalitis virus through formation of covalent cross-link has also been observed and the cross-link was partially identified by MS/MS analysis. Thus, the squaramate-linked CTP is a useful building block for the synthesis of reactive RNA probes for bioconjugations with amines and cross-linking with lysine residues.