# RNA-Peptide Conjugation through an Efficient Covalent Bond Formation
## 4 Discussion  
A Schiff base formation between the hydraz and aldehyde group rapidly progressed at a mild acidic condition although 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 the wide range of pH helped us to understand a limitation and the applicability of this reaction under the physiological condition for RNA-protein or RNA-peptide conjugation. Formation of the covalent linkage between RNA and the Rev peptide subunit in the Rev-RRE complex quantitatively proceeded in a mild acidic to a neutral pH condition within 3 h. The result indicated the versatility of this reaction in a physiological condition for the crosslink formation of peptide and RNA. Unlike the sequence-specific nature for the stable noncovalent RNP complex formation, formation of the covalent linkage between the 3\({}^{\prime}\)-dialdehydedebyde group of RNA and the hydrazide group at the C-terminal of Rev peptide required no specific RNA sequence for the RNA binding of Rev peptide. Even the nonspecific RNA-peptide complex formation driven by the electrostatic interaction was sufficient for exerting the proximity effect of reactive groups, thereby resulting an efficient formation of covalent linkage between the Rev peptide and RNA. The significant decrease of the reaction efficiency by deletion of the cationic moiety in the peptide sequence also showed the contribution of the electrostatic interaction for the efficient proceeding of the crosslinking reaction. We designed a DNA sequence-specific protein-tag that underwent a proximity-driven intermolecular crosslinking between protein and DNA [43; 44; 45; 46; 47; 48; 49]. Selective DNA modification by a self-ligating protein tag conjugated with a DNA-binding domain, termed as a modular adaptor, was achieved by relying on the chemoselectivity of the protein tag [50; 51; 52]. By tuning the alkylation kinetics for the protein-tag and its substrate, the sequence-specific crosslinking reaction of the modular adaptor was exclusively driven by the DNA recognition when the dissociation rate of the DNA complex was much larger than the rate constant for the alkylation reaction [45; 46; 47; 48; 49]. In connection with these findings, a sequence-specific crosslinking reaction between the 3\({}^{\prime}\)-dialdehydedebyde group of RNA and the hydrazide group of RNA-binding peptide would be realized by tuning the reaction conditions, such as the concentration of sodium salt. As we have reported previously, the crosslinking reaction between the 3\({}^{\prime}\)-dialdehydedebyde group of RNA and the hydrazide group of peptide was applied for the facile construction  
Figure 4: A denaturing PAGE (8 M Urea) analysis of the conjugation reaction products of c-RNP constructed from An16 and Ac-(GGS)\({}_{3}\)G-HZ. Lane 1: An16 RNA; Lane 2: c-An16/Ac-Rev-(GGS)\({}_{3}\)G-HZ reacted for 3 h; Lane 3: c-An16/Ac-(GGS)\({}_{3}\)G-HZ reacted for 14 h.  
of covalently crosslinked fluorescent RNP sensors [41; 42]. The covalently crosslinked fluorescent RNP sensor not only realized an improved stability of RNP sensors but also expand its application, such as the simultaneous detection of multiple targets in the solution. The noncovalent RNP scaffold was effective for the library-based selection and the cooperative functionalization of RNP receptor, such as the fluorescent sensors and catalysts [53]. Once a functional noncovalent RNP was obtained, formation of a covalent linkage within the RNA-peptide complex provided stable RNP for a wide range of applications. A natural RNA oligonucleotide and facile preparation of the reactive peptide was only needed to perform this reaction. Our investigations regarding the effect of the pH, RNA, and a peptide sequence for the reaction efficiencies in this reaction would help us to understand the limitation and applicability of this method for construction of RNA-peptide or protein conjugate.