Abstract
Many methods for modification of an oligonucleotide with a peptide have been developed to apply for the therapeutic and diagnostic applications or for the assembly of nanostructure. We have developed a method for the construction of receptor-based fluorescent sensors and catalysts using the ribonucleopeptide (RNP) as a scaffold. Formation of a covalent linkage between the RNA and the peptide subunit of RNP improved its stability, thereby expanding the application of functional RNPs. A representative method was applied for the formation of Schiff base or dihydroxy-morpholino linkage between a dialdehyde group at the 3′-end of sugar-oxidized RNA and a hydrazide group introduced at the C-terminal of a peptide subunit through a flexible peptide linker. In this report, we investigated effects of the solution pH and contribution of the RNA and peptide subunits to the conjugation reaction by using RNA and peptide mutants. The reaction yield reached 90% at a wide range of solution pH with reaction within 3 h. The efficient reaction was mainly supported by the electrostatic interaction between the RNA subunit and the cationic peptide subunit of the RNP scaffold. Formation of the RNP complex was verified to efficiently promote the reaction for construction of the RNA-peptide conjugate.
Highlights
RNA and peptide conjugates have been constructed for versatile therapeutic application, such as the delivery of siRNA [1,2,3] or the screening of a library of peptides in a mRNA display method [4,5].Efficient reaction for the formation of covalent linkage between RNA and peptide is important for developing practical applications
A Schiff base formation between the hydrazide and aldehyde group rapidly progressed at a mild mild acidic condition the stability was less than that at a neutral pH
Because the reaction efficiency of this crosslinking method was based on the balance of such properties, the assays under efficiency of this crosslinking method was based on the balance of such properties, the assays under the wide range of pH helped us to understand a limitation and the applicability of this reaction the wide range of pH helped us to understand a limitation and the applicability of this reaction under under the physiological condition for RNA-protein or RNA-peptide conjugation
Summary
RNA and peptide conjugates have been constructed for versatile therapeutic application, such as the delivery of siRNA [1,2,3] or the screening of a library of peptides in a mRNA display method [4,5]. By coupling the aldehyde group with alkoxyamine, cysteine, and hydrazine, formation of the covalent linkage through oxime, thiazolidine, and hydrazone (or a morpholine-like structure), respectively, has been reported [27,28,29] These reactions proceed in mild conditions in a relatively short time to provide high coupling yields in aqueous solution. Coupling of the RNA dialdehyde group and the C-terminal hydrazide group of peptide formed a covalent linkage between the RNA and peptide subunits of the Rev-RRE complex This reaction proceeded rapidly in a quantitative yield. Our results demonstrated that the efficient coupling reaction within the RNP was the outcome of the proximity effect of reactive groups and provided an optimal condition for the construction of the covalently linked RNA-peptide complex.
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