# RNA-Peptide Conjugation through an Efficient Covalent Bond Formation  
Shun Nakano  
Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan; [EMAIL_ADDRESS] (S.N.); [EMAIL_ADDRESS] (T.S.); [EMAIL_ADDRESS] (Z.Z.)  
12 October 20202010 December 2  
rather low. One of the alternative methods for the modification of nucleotide with functional molecules was based on incorporating the aldehyde group into the nucleotide. Oxidation of the sugar moiety by perchloric acid or periodate is one of the common methods for introducing the aldehyde group on DNA or RNA [24; 25; 26; 27]. By coupling the aldehyde group with alkoxyamine, cysteine, and hydrazine, formation of the covalent linkage through oxime, thiazolline, 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. While this strategy also displays some of the limitations, as mentioned above, it has the advantage of having no need for the introduction of unnatural nucleic acids into oligonucleotide and facile preparation of the reactive peptide.  
We have developed a stepwise method for the construction of receptor-based fluorescent sensors by using ribonucleopeptide (RNP) as a scaffold (Figure 1A). In this method, a complex of Rev peptide and Rev Responsive Element (RRE) RNA [30] was utilized to construct the RNP library with a randomized RNA sequence in the RNA subunit. RNP receptors [31] for a target molecule were selected from the RNP library by applying the in vitro selection method [32; 33]. The peptide subunit of the selected RNP receptors was further modified with a fluorophore to construct a fluorophore-modified RNP receptor (F-RNP) library [34; 35; 36; 37; 38; 39; 40; 41; 42]. By screening the F-RNP library, fluorescent RNP sensors showing measurable fluorescent intensity changes upon binding the substrate were selected. Many kinds of RNP receptors and sensors were constructed for various target molecules, such as ATP [34; 35; 36; 37; 38; 41; 42], GTP [34; 35; 41], dopamine [39], and a tetra peptide containing a phosphorylated tyrosine residue [40]. Furthermore, the RNA subunit and the peptide subunit of fluorescent RNP sensors were covalently linked to improve the chemical and thermal stability [41]. The covalently linked RNP (c-RNP) sensors were applicable for simultaneous detection of multiple target molecules in the solution. Time-course monitoring of the concentration changes of the substrate and product in an enzymatic reaction was demonstrated by simultaneous application of sensors for the substrate and the product [41; 42]. As mentioned above, for the covalent bond formation with the aldehyde group, the ribose moiety at the 3\({}^{\prime}\)-end of RNA was oxidized by sodium periodate to a dialdehyde group (Figure 1B). A hydrazide group was introduced at the C-terminal of peptide subunit through a ten-amino-acid flexible linker. 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. It is likely that a proximity effect between the reactive groups on the RNA and peptide subunit originated from the complex formation of RNP to assist the efficient coupling reaction. Here, we investigated several conditions for covalent linkage formation between the dialdehyde group of RNA and the hydrazide group of peptide within the ribonucleopeptide scaffold to elucidate the effect of solution pH for the reaction yield. Furthermore, the role of proximity effect in the coupling reaction was investigated by using mutants of RNA and peptide. Our results demonstrated that the efficient coupling reaction within the RNP was indeed the outcome of the proximity effect of reactive groups and provided an optimal condition for the construction of the covalently linked RNA-peptide complex.