RNA-Peptide Conjugation through an E ﬃ cient Covalent Bond Formation

: 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 sca ﬀ old. 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 Schi ﬀ base or dihydroxy-morpholino linkage between a dialdehyde group at the 3 (cid:48) -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 e ﬀ ects 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 e ﬃ cient reaction was mainly supported by the electrostatic interaction between the RNA subunit and the cationic peptide subunit of the RNP sca ﬀ old. Formation of the RNP complex was veriﬁed to e ﬃ ciently promote the reaction for construction of the RNA-peptide conjugate. e ﬃ cient production of RNA-peptide


Introduction
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. Many methods have been reported to conjugate an oligonucleotide (RNA or DNA) with an oligopeptide by applying various chemistries [6][7][8][9]. Post-synthetic coupling of the respective oligonucleotide and peptide fragments is the common and reliable method to construct oligonucleotide-peptide conjugates because the stepwise solid-phase synthesis of the conjugates encounters difficulties in finding the compatible protecting groups for both nucleobases and amino-acid side chains. Several chemical reactions were applied for coupling the nucleotide and peptide, such as amidation [10,11], disulfide bond formation [12][13][14], the modification of the thiol group by haloacetyl [15,16], or the maleimide group [12,17,18], native chemical ligation [19][20][21], and click reaction [22,23]. These reactions are certainly useful for the selective coupling of oligonucleotides and oligopeptides. However, these reactions often associate with drawbacks, such as the instability of the linkage, the necessity for introducing additional chemical groups into both the oligonucleotide and the oligopeptide chains by multistep reactions, limitation for the sequence of oligopeptide due to the solubility or reactivity of the side chains, and/or the potential formation of the side products and the stereoisomers. In some cases, even applying the long reaction time, the yields of the product were

Preparation of RNA
Forward and Reverse primers for the construction of the double-stranded template DNA for An16 RNA (5′-TCTAATACGACTCACTATAGGTCTGGGCGCA-3′ and 5′-GGCCTGTACCGTC-3′) and for scrAn16 RNA (5′-TCTAATACGACTCACTATAGGAGGCTTCAGCTTCG-3′ and 5′-CACACAACCGCCCCG-3′) were purchased from Sigma-Aldrich, Japan (T7 RNA promoter is underlined). The double-stranded DNA templates and RNAs were prepared as previously described [42]. RNA subunits of RNP receptors were purified by means of denaturing polyacrylamide gel Schematic illustration for the construction of the covalently linked fluorescent ribonucleopeptide (RNP) sensor. RNA receptors selected from the RNA-derived RNP library were converted into a fluorescent RNP sensor library through modification of the peptide subunit by a fluorophore. (A) A fluorescent RNP sensor, selected from the fluorescent RNP sensor library, was converted to a covalently linked RNP (c-RNP) sensor by the crosslinking reaction between RNA and the peptide subunit [41]. (B) Scheme of covalent linkage formation between RNA and the peptide subunit of RNP.

Construction of a Covalently Linked RNP
The peptide subunit for the formation of a covalent linkage (Ac-TRQARRNRRRRWRERQR-GGSGGSGGSG-HZ) was synthesized as follows. A HMBA-PEG resin was placed in a dry flask, and a sufficient amount of DMF was added to soak the resin; this mixture was allowed to swell for 30 min. N-α-Fmoc-glycine (10 equivalent relative to resin loading) dissolved in DMF was mixed with a solution of DIC (5 equivalent relative to resin loading) in dry DMF on ice and then incubated for 20 min. The solution of an activated first amino acid was added to the resin prepared above. A DMF solution of DMAP (0.1 equivalent relative to resin loading) was added to the resin/amino acid mixture and incubated at room temperature for 1 h with occasional swirling. This procedure of coupling the first amino acid residue was repeated twice. The subsequent synthesis was performed on an automated peptide synthesizer (PSSM-8; Shimadzu, Kyoto, Japan) according to the Fmoc chemistry protocol using protected Fmoc-amino acids and HBTU. Acetylation of the N-terminal of peptide was performed by mixing with 1 M acetic acid anhydride and 1 M 1-methyl imidazole in DMF for 1 h at room temperature. The following procedures, cleavage of the protected peptide from the resin with hydrazine monohydrate, deprotection of the protected peptide, and purification of the peptide were performed as described previously [41]. The synthesized peptides were characterized by MALDI-TOF mass spectrometry (AXIMA-LNR, Shimadzu), as follows: Acetylated Rev peptide hydrazide (Ac-Rev-(GGS) 3  Crosslinking reaction of the RNA and the peptide subunits of RNP was carried out as described previously [41,42] with a slight modification of the conditions. Freshly prepared 0.01 M sodium periodate (5 µL; 50 nmol; 50 equiv) was added to 200 µM RNA (5 µL; 1 nmol) in 15 µL of 0.03 M sodium acetate (pH 5.2), and the reaction mixture (25 µL) was incubated for 1 h at 37 • C in the dark. After the reaction, 2.5 µL of 10 M glycerol was added to the reaction mixture to reduce an excess amount of sodium periodate. The resulting oxidized RNA was purified by ethanol precipitation. A coupling reaction between the 3 -modified RNA (40 µM) and Ac-Rev-(GGS) 3 G-HZ (88 µM) was performed in 0.03 M sodium acetate (for pH 4 and 5) or 0.03 M sodium phosphate (for pH 6 and 7), containing 0.01 M NaCl (total 25 µL) at 37 • C in the dark. The reaction mixture was extracted by phenol/chloroform and purified by ethanol precipitation to remove unreacted peptide, then dissolved in TE (25 µL).

Evaluation of the Reaction Yields of Covalently-Linked RNPs
Denaturing polyacrylamide gel electrophoresis (PAGE) (8 M urea, 12%) was performed to separate the unreacted RNA and covalently linked RNP by loading the same volume of the purified samples. A total of 10 pmol of RNA was loaded on the gel as the control of RNA band mobility. The above stock solution of phenol/chloroform extracted RNP (25 µL) was further diluted with TE to 200 µL. From this RNP solution, 2 µL was analyzed by PAGE for each reaction. The acrylamide gel was stained with ethidium bromide to detect RNA and RNP. The yield of each reaction was evaluated from the ratio of intensity of bands corresponding to unreacted RNA and RNP. Actual isolation yields of RNP ranged from 30 to 40% after the PAGE purification and successive purification by ethanol precipitation.

MALDI-TOF Mass Analysis of the Reaction Solution
The reaction solution removed the unreacted peptide by phenol/chloroform extraction, and ethanol precipitation was characterized by MALDI-TOF mass spectrometry (AXIMA-Confidence, Shimadzu, Kyoto, Japan). A matrix solution was prepared as a mixture (v/v: 9/1) of 10 mg/mL of THAP in acetonitrile/H 2 O (v/v: 1/1) and 50 mg/mL of DAHC in pure water. The sample solution was mixed with the same volume of matrix solution (1 µL each). DNA oligonucleotides (molecular weight: 5828.9, 11,397.5, 13,976.2, 23,164.3) were used as the external calibration standard sample. All measurements were performed in linear positive mode.

Effect of Solution pH for the Formation of Covalent Linkage
The effect of solution pH for the formation of covalent linkage between the 3 -dialdehyde group of the oxidized RNA subunit and the hydrazide group at the C-terminal of peptide subunit of the Rev-RRE complex was investigated by carrying out the reaction at various pH levels. The RNA subunit of ATP-binding of the RNP receptor, An16, was oxidized by using sodium periodate in a sodium acetate buffer at pH 5. The reaction was performed at 37 • C for 1 h. After isolation of the oxidized RNA by ethanol precipitation, acetylated Rev peptide, modified with a C-terminal hydrazide group through a flexible linker (GGS) 3 G (Ac-Rev-(GGS) 3 G-HZ), was mixed with the oxidized RNA in sodium acetate buffer at pH 4 or 5 or in a sodium phosphate buffer at pH 6 or 7. The reaction mixtures were incubated for 3 h at 37 • C, then the reactions were stopped by the addition of phenol/chloroform and vigorously mixed. The water layer was condensed by ethanol precipitation. The reaction yield of c-RNP in each solution was evaluated by quantitation of the band intensity, corresponding to free RNA and conjugated c-RNP in a denaturing polyacrylamide gel electrophoresis (PAGE) (Figure 2). Over 80% of RNA was reacted with peptide in all conditions. The yields of reaction were 92 ± 4% at pH 4, 92 ± 5% at pH 5, 86 ± 3% at pH 6, and 82 ± 3% at pH 7 ( Figure S1A). These results indicated that the solution pH ranging from 4 to 7 did not affect much to the yield of RNA-peptide conjugate. Prolonged incubation over 15 h at pH 5 did not show any difference in the ratio of the band intensity for c-RNP and unreacted RNA (data not shown).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 10 weight: 5828.9, 11,397.5, 13,976.2, 23,164.3) were used as the external calibration standard sample. All measurements were performed in linear positive mode.

Effect of Solution pH for the Formation of Covalent Linkage
The effect of solution pH for the formation of covalent linkage between the 3′-dialdehyde group of the oxidized RNA subunit and the hydrazide group at the C-terminal of peptide subunit of the Rev-RRE complex was investigated by carrying out the reaction at various pH levels. The RNA subunit of ATP-binding of the RNP receptor, An16, was oxidized by using sodium periodate in a sodium acetate buffer at pH 5. The reaction was performed at 37 °C for 1 h. After isolation of the oxidized RNA by ethanol precipitation, acetylated Rev peptide, modified with a C-terminal hydrazide group through a flexible linker (GGS)3G (Ac-Rev-(GGS)3G-HZ), was mixed with the oxidized RNA in sodium acetate buffer at pH 4 or 5 or in a sodium phosphate buffer at pH 6 or 7. The reaction mixtures were incubated for 3 h at 37 °C, then the reactions were stopped by the addition of phenol/chloroform and vigorously mixed. The water layer was condensed by ethanol precipitation. The reaction yield of c-RNP in each solution was evaluated by quantitation of the band intensity, corresponding to free RNA and conjugated c-RNP in a denaturing polyacrylamide gel electrophoresis (PAGE) (Figure 2). Over 80% of RNA was reacted with peptide in all conditions. The yields of reaction were 92 ± 4% at pH 4, 92 ± 5% at pH 5, 86 ± 3% at pH 6, and 82 ± 3% at pH 7 ( Figure  S1A). These results indicated that the solution pH ranging from 4 to 7 did not affect much to the yield of RNA-peptide conjugate. Prolonged incubation over 15 h at pH 5 did not show any difference in the ratio of the band intensity for c-RNP and unreacted RNA (data not shown).

Contibution of the RNA-Peptide Interaction for the Reaction Yield
RNA sequence dependency of the reaction was next evaluated to address the proximity effect of reactive groups. The specific interaction between the Rev peptide and RRE RNA of the RNP scaffold was expected to have an influence on the efficiency of covalent linkage formation. A sequencescrambled scrAn16 RNA that contained the same number of each nucleotide to An16 RNA was

Contibution of the RNA-Peptide Interaction for the Reaction Yield
RNA sequence dependency of the reaction was next evaluated to address the proximity effect of reactive groups. The specific interaction between the Rev peptide and RRE RNA of the RNP scaffold was expected to have an influence on the efficiency of covalent linkage formation. A sequence-scrambled scrAn16 RNA that contained the same number of each nucleotide to An16 RNA was prepared ( Figure 3A). The reaction of the oxidized scrAn16 with Ac-Rev-(GGS) 3 G-HZ was performed at pH 5. The MALDI TOF MS analysis using the reaction solution after removing the unreacted peptide was performed to characterize the products. The production of covalently linked RNP was confirmed by observing respective MS peaks (Figures S2 and S3). In the five trials, scrAn16 showed a similar averaged reaction yield (92 ± 5%) to that of the parent An16 (92 ± 5%) when conjugated with the hydrazide group of Ac-Rev-(GGS) 3 G-HZ ( Figure 3B and Figure S1B,D). The result showed that the RNA sequence did not affect the efficiency of reaction. The non-specific electrostatic interaction between the RNA and the cationic Rev peptide would relate to the reaction efficiency. To verify this notion, the reaction for the formation of covalently linked RNP was carried out by using a truncated peptide of Ac-Rev-(GGS) 3 G-HZ. A truncated peptide possessing only the GGS peptide linker moiety, Ac-GGSGGSGGSG-HZ, was designed by deleting the Rev peptide sequence from Ac-Rev-(GGS) 3 G-HZ. The truncated peptide showed a significant decrease in the yield to 57 ± 1%, even after a prolonged reaction time for 14 h (Figure 4 and Figure S1C). This result supported the notion that the electrostatic interaction between RNA and the peptide mainly contributed to increasing the reaction yield. Thus, the RNP scaffold was useful for the efficient production of RNA-peptide conjugates.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 10 prepared ( Figure 3A). The reaction of the oxidized scrAn16 with Ac-Rev-(GGS)3G-HZ was performed at pH 5. The MALDI TOF MS analysis using the reaction solution after removing the unreacted peptide was performed to characterize the products. The production of covalently linked RNP was confirmed by observing respective MS peaks (Figures S2 and S3). In the five trials, scrAn16 showed a similar averaged reaction yield (92 ± 5%) to that of the parent An16 (92 ± 5%) when conjugated with the hydrazide group of Ac-Rev-(GGS)3G-HZ ( Figure 3B and Figure S1B,D). The result showed that the RNA sequence did not affect the efficiency of reaction. The non-specific electrostatic interaction between the RNA and the cationic Rev peptide would relate to the reaction efficiency. To verify this notion, the reaction for the formation of covalently linked RNP was carried out by using a truncated peptide of Ac-Rev-(GGS)3G-HZ. A truncated peptide possessing only the GGS peptide linker moiety, Ac-GGSGGSGGSG-HZ, was designed by deleting the Rev peptide sequence from Ac-Rev-(GGS)3G-HZ. The truncated peptide showed a significant decrease in the yield to 57 ± 1%, even after a prolonged reaction time for 14 h (Figure 4 and Figure S1C). This result supported the notion that the electrostatic interaction between RNA and the peptide mainly contributed to increasing the reaction yield. Thus, the RNP scaffold was useful for the efficient production of RNA-peptide conjugates.

Discussion
A Schiff base formation between the hydrazide 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′-dialdehyde 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 selfligating 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′-dialdehyde 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′-dialdehyde group of RNA and the hydrazide group of peptide was applied for the facile construction of covalently crosslinked fluorescent RNP sensors [41,42]. The covalently crosslinked fluorescent RNP sensor not only realized an improved stability of RNP sensors

Discussion
A Schiff base formation between the hydrazide 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 -dialdehyde 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 -dialdehyde 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 -dialdehyde group of RNA and the hydrazide group of peptide was applied for the facile construction 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. It would also be helpful for the development of an emerging application of oligonucleotide-peptide conjugates, such as a self-assembled scaffold of nanostructure or an efficient delivery system of functional RNA into the cell.

Conflicts of Interest:
The authors declare no conflict of interest.