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

doi:10.21203/rs.3.rs-4705129/v1

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

https://doi.org/10.21203/rs.3.rs-4705129/v1

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We designed and synthesized cytidine 5′-O-triphosphate bearing a reactive squaramate group attached to position 5 of pyrimidine (C^ESQTP) 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.

Physical sciences/Chemistry/Chemical biology/Nucleic acids

Physical sciences/Chemistry/Biochemistry/RNA

RNA-protein interactions are a very important yet underexplored field of research.¹ One of the most efficient ways how to study them is the use of covalent cross-linking of RNA with interacting proteins.^2,3,4 So far, the common methods include photochemical cross-linking^5,6,7,8 or usage of external chemical cross-linking agents.^9,10,11 These methods are often inefficient, non-specific and toxic for the use in live cells. Therefore, there is a need for introduction of specific reactive groups into RNA for cross-linking with peptides and proteins. In related DNA, there are many established reactive groups that can be linked to nucleobases in 2’-deoxyribonucleoside triphopshates (dNTPs) and incorporated enzymatically,¹² e.g. Michael acceptors reacting with Cys,¹³ chloroacetamide reacting with Cys or His,¹⁴ and glyoxal¹⁵ or 1,3-diketones¹⁶ for Arg, as well as nitrosobenzaldehyde¹⁷ or squaramate¹⁸ for Lys. On the other hand, there were only few examples of protein-cross-linking modifications of RNA relying on 5-fluoro- or 5-azapyrimidine nucleotides that form covalent adducts with Cys-containing proteins, e.g. methyltransferases.^19,20,21 Other related examples of reactive modifications of RNA included posttranscriptional labelling through bioorthogonal reactions^{22,23,24,25,26,27,28,29} or alkylation of RNA with methyltransferases.^30,31,32,33 Recently, we reported chloroacetamide-linked ATP that can be incorporated to RNA by in vitro transcription (IVT) using T7 RNA polymerase (T7 RNAP) and cross-links to Cys- or His-containing peptides and proteins,³⁴ but there is an urgent need for reactive RNA probes specific for Lys as one of the most abundant amino acid in nucleic acids binding proteins.

The well established IVT with T7 RNAP using modified ribonucleoside triphosphates (NTPs) incorporates the modifications replacing natural counterparts at all sites.²³^-²⁵^,35,36,37 Enzymatic site-specific single modification with RNA polymerases is very complicated³⁸ and was previously reported only through laborious methods of limited efficiency, i.e. position-selective labeling by T7 RNAP (PLOR),³⁹ splint ligation⁴⁰ or use of extended genetic alphabet.²⁶^-²⁹ Only very recently we⁴¹ and others⁴² have independently reported enzymatic synthesis of base-modified RNA using engineered TGK DNA polymerase and DNA template for extension of an RNA primer (PEX) and an expedient site-specific or segmented incorporation of one or more modifications through (repeated) single nucleotide incorporation followed by primer extension. This approach takes advantage of the high substrate tolerance and efficiency of DNA polymerases for incorporation of even highly modified NTPs and of the possibility to exchange single-stranded DNA template to synthesize specifically modified RNA not accessible by classical IVT.

In this paper, we present the design and development of Lys-reactive RNA probes through enzymatic synthesis by incorporation of the corresponding squaramate-modified ribonucleoside triphosphate (rNTP) with T7 RNAP or TGK DNA polymerase and applications in bioconjugations and cross-linking with Lys-containing peptides and proteins and studying and inhibition of viral RNA polymerases.

Synthesis of modified nucleoside and C^ESQTP

We designed the squaramate-modified ribonucleoside triphosphate in similar way to the previously reported dNTP.¹⁸ The squaramate was linked to position 5 of cytosine through propargyl amide linker to ensure good compatibility with polymerase incorporation. The synthesis (Fig. S1 in SI and Fig. 2A) started by the Sonogashira reaction of 5-iodocytidine with N-propargyl trifluoroacetamide followed by deacylation to give 5-(3-aminopropyn-1-yl)-cytidine 3 in very good yield (87%). The reaction of nucleoside 3 with 2 equiv. of diethyl squarate gave squaramate-linked ribonucleoside (C^ESQ) in acceptable yield (22%). The triphosphorylation⁴³ of C^ESQ with POCl₃ in trimethyl phosphate followed by treating with tributyl ammonium pyrophosphate gave the corresponding C^ESQTP (38%).

RNA synthesis by in vitro transcription with C^ESQTP and T7 RNAP

We tested the ability of C^ESQTP to serve as a CTP analog for RNA polymerase in IVT (Fig. 2B). We performed the IVT reactions in the presence of various DNA templates (for sequences of ONs, see Supplementary Table S1). The templates contained one, three, seven or eight dG nucleotides in the coding region that guided the incorporation of one to eith of C^ESQ nucleotide into RNA transcript. We used two 5'-terminal 2'-MeO ribonucleotides of antisense strand of DNA templates to minimize non-templated nucleotide addition⁴⁴ and the IVT reactions were performed in the presence of T7 RNA polymerase, ATP, UTP, GTP, CTP/modified CTP (C^ESQTP) and spiked with radioactive [α-³²P]-GTP. The transcription conditions needed to be optimized to avoid the undesired reaction of newly synthesized squaramate modified RNA with spermidine from commercial transcription buffer supplied with polymerase (Supplementary Fig. S3a, S3c and S17). Squaramate moiety is known to prefer primary amines to the more substituted ones,⁴⁵ and hence to overcome this undesired reaction, spermidine was replaced with quarternary amine betaine. All transcription reactions were analysed on an analytical polyacrylamide gel and phosphor-imaged (Fig. 2C, for uncropped gels see Supplementary Fig. S2). The full-length RNA products were also characterized by MALDI-TOF (Supplementary Fig. S18-24). Additional control analysis of reaction mixture on both native and denaturing protein gels excluded crosslinking of squaramate modified RNA with the T7 RNA polymerase during transcription reaction (Supplementary Fig. S3e and S9). The IVT synthesis of RNA containing one or three C^ESQ modifications (i.e. 35_RNA_1C^ESQ or 35_RNA_3C^ESQ) proceeded smoothly with good conversions, whereas the formation of RNA with high number of reactive modifications (i.e. 35_RNA_7C^ESQ) was less efficient (Fig. 2C).

RNA synthesis by PEX with C^ESQTP and TGK polymerase

Following our recent paper on enzymatic synthesis of base-modified RNA using engineered DNA polymerases,⁴¹ we tested C^ESQTP as a substrate for RNA synthesis by PEX (Fig. 2D) using TGK polymerase.⁴⁶ We used a 31-nt single-stranded DNA template encoding for four cytosines in the complementary strand and a 5’-FAM-labeled 15-nt RNA primer. The PEX reaction was followed by digesting template by DNase, and we analyzed the products by PAGE (Fig. 2E and Supplementary Fig. S5). The formation of the desired modified RNA FAM_31RNA_4C^ESQ containing four modified C^ESQ nucleotides was confirmed by PAGE and LC-MS (Supplementary Fig. S5). MS analysis also revealed a non-templated addition of GMP resulting in one nucleotide longer RNA.

Conjugation reactions of ESQ-modified RNA with an amine and Lys-containing peptide

First, we tested the reactivity of C^ESQ-modified RNA with a primary amine. We used amine-linked sulfo-Cy5-amine (Fig. 3A, Supplementary Fig. S6) for site-specific RNA labelling. Non-labelled natural RNA (for control experiment) and squaramate-modified RNA (35_RNA_1C^ESQ) was prepared by in vitro transcription followed by DNase I treatment to digest the DNA templates and purified by spin column method. Model cross-linking reaction was performed with sulfo-Cy5-amine (100 - 400 equiv.) in buffer (pH 7.4-7.9) at 37°C. After overnight incubation, the Cy-5 labelled RNA was purified to get rid of the excess dye and analyzed by fluorescence spectroscopy and by fluorescence imaging of the denaturing gel. Strong fluorescence signal was observed only for modified RNA while the non-modified RNA was not labeled under the same conditions (Fig. 3B, Supplementary Fig. S6a). The fluorescence imaging of the denaturing gel, shown in Supplementary Fig. S6b, supports the conclusion of fluorescence measurements that a stable covalent conjugate of Cy5 with RNA (35_RNA_1C^ESQ_Cy5) has been formed.

Then we tested the reactions of ESQ-modified RNAs with a Lys-containing peptide Ac-Lys-Ala-Ala. We used 35_RNA_1C^ESQ and 35_RNA_3C^ESQ prepared by in vitro transcription using T7 RNA polymerase followed by DNase I treatment, purified and characterized by dPAGE (Supplementary Fig. S4) and LCMS (Supplementary Fig. S28 and S29). Using 35_RNA_3C^ESQ containing 3 reactive modifications, we initially tested influence of peptide excess (500 or 5000 equiv.) in borate buffer of different pH (9 or 10) for RNA-peptide conjugation efficacy. The buffer pH did not show any significant effect to the conjugation performance. On the other hand, increased excess of peptide pushed the conversion towards RNA-peptide conjugate but despite of that, we still observed mixture of RNA-peptide conjugates with two and three peptide molecules attached (Supplementary Fig. S7). Having different electrophoretic mobilities, we could identify them on dPAGE and confirm by LC-MS (Supplementary Fig. S32). We then used the higher peptide excess (9000 equiv.) in two different buffer systems (borate buffer pH 9 and carbonate-bicarbonate buffer, pH 9.5) for the conjugation of 35_RNA_1C^ESQ (Fig 3D) to yield the corresponding covalent conjugate 35_RNA_1C^ESQ_KAA in 80 and 75 % conversions, respectively. The conjugation of 35_RNA_3C^ESQ gave mixture of several conjugates containing one or more peptide moieties (Supplementary Fig. S8 and S30).

Cross-linking of RNA_C^ESQ with proteins

For cross-linking reactions of the ESQ-modified RNA with RNA binding proteins (RBP) we selected three RNA dependent RNA polymerases (RdRp; Japanese encephalitis virus (JEV) NS5 protein, Yellow fever virus (YFV) NS5⁴⁷ and SARS-CoV-2 (SC_RdRp) RdRp,⁴⁸ as well as SARS-CoV-2 nucleoprotein (SC_NP)⁴⁹ and HIV reverse transcriptase (HIV-rt)⁵⁰ as a biologicaly relevant targets each of them containing several Lys amino acid residues. Bovine serum albumin (BSA) an RNA non-binding protein was used as a negative control and Single strand binding protein as a DNA binding protein with 10-fold lower affinity for ssRNA.⁵¹ For all RNA-protein crosslinking studies, radioactively labeled natural or modified RNA was used (prepared by IVT reaction described in methods section). RNA-protein cross-linking reactions were performed at pH 7.4-8, with 2 equiv. of HIV-rt, 4 equiv. of JEV and YFV NS5 protein, 8 equiv. of SC_RdRp and SC_NP and higher excess of RNA non-binding or RNA weakly binding proteins (20 equiv. of BSA and SSB). The ability of proteins to bind to the 36_vRNA_1gC^ESQ (bearing one modification; for sequence see Supplementary Table S1) target was monitored by electrophoretic mobility shift assay (EMSA). Reaction analysis by 10% native PAGE confirmed that the squaramate modification did not prevent protein binding. In all cases a band with slower mobility was observed, although the complex formation between RNA and SC_RdRp was very weak (Fig. 4b). As expected, no protein-RNA complex was formed in the case of BSA and T7 RNApol (Supplementary Fig. S9 and S10). The denaturing SDS-PAGE revealed formation of corresponding RNA-protein covalent cross-links represented by the bands with slower mobility (RNA^ESQ_SSB9%, RNA^ESQ_HIV_rt 15%, RNA^ESQ_JEV_NS543%, RNA^ESQ_YFV_NS540%; Fig. 2b, Supplementary Fig. S10, S11 and S14, Table 1). Only traces of covalent cross-link were detected for 36_vRNA^ESQ_SC_RdRp and 36_vRNA^ESQ_SC_NP (Fig. 2b, Supplementary Fig. S13, Table 1). More modifications in the RNA strand in case of 36_vRNA_3gC^ESQ did not lead to better RNA-protein binding, nor better covalent cross-linking coversions (Supplementary Fig. S11 and S13, Table S3). Moreover, the covalent RNA^ESQ_SSB conjugate was confirmed by ESI-MS analysis (Supplementary Fig. S33).

Table 1 RNA-protein cross-link conversions between 36_vRNA_1gC^ESQ and recombinant proteins.

Protein	Molecular weight [kDa]	RNA binding	No of Lys	Conversion %
BSA	69.3	no	60	0%
T7 RNAP	99	no	66	0%
SSB	18.9	weakly	6	9%
HIV rt	117	yes	108	15%
JEV NS5	103.3	yes	58	43%
YFV NS5	103.3	yes	60	40%
SC RdRp	62.2	yes	72	traces
SC NP	45.6	yes	31	traces

Note: Conversions were determined from the gel using ImageJ Quantificator.

Gel-based RdRp polymerase assays in vitro

Following the cross-linking studies that have shown that the RdRps from Flaviviridae family readily cross-linked with squaramate modified RNA, we decided to investigate how modified triphosphate (C^ESQTP) influences the polymerase activity of the JEV recombinant RdRp (JEV NS5) and SARS-CoV-2 RdRp (SC RdRp) on an RNA template-product scaffold in vitro (Fig. 5a and Supplementary Fig. S15 and S16). We used gel based RdRp polymerase assay conditions similar to those published for various flaviviruses⁵² and non-modified templates that represent the 3'-end of JEV genome of the c5'-UTR, that fold into a hairpin with 5'-end FAM labelled overhang as a template for primer extension (for sequences see Supplementary Table 2).⁵³ RdRp polymerases are known to go through repeated cycles of abortive initiation before starting elongation mode⁵⁴ and consecutive cytosines in the template may cause polymerase slippage⁵⁵ which leads to the presence of short abortive products and ladder-like pattern on the gel. SARS-CoV-2 RdRp produced full length (ESQ)-modified RNA, which was confirmed both by analytical polyacrylamide gel (Supplementary Fig. S16a-b) and MALDI (Supplementary Fig. S25-26). On the other hand, in the case of the JEV recombinant RdRp, we observed the full-length product of natural RNA and only traces of squaramate modified RNA (Supplementary Fig. S15a-b). After subsequent treatment of the reaction mixture with Proteinase K (pK) to release all RNA from JEV NS5 protein-RNA complex, both natural and modified full-length RNAs were observed with additional band of slower mobility in the 46_RNA_HP^ESQ lane (Fig. 3b, lanes 3 and 4 respectively; Supplementary Fig. S15a-b). The intensity of this new band increased after prolonged reaction time (Fig. 5b, lanes 5 and 6 respectively; Supplementary Fig. S15a-b). We assumed that this band might be the squaramate modified RNA cross-linked to the short peptide resulting from JEV NS5 protein digestion. Additionally, the presence of cross-linked product of new modified RNA and full-length JEV NS5 RdRp was independently proved by comparison of 10% SDS protein gel visualized both by FAM for RNA and immunodetection of JEV NS5 (Fig. 3c; Supplementary Fig. S15d-e). These results suggest that the JEV RdRp incorporates C^ESQTP and subsequently cross-links with the produced modified RNA during the polymerase reaction.

Proteomic analysis of 46_RNA_HP^C4-JEV conjugate

To characterize cross-linked product fully and to determine the RNA-protein binding sites, two independent samples of the reaction mixture after polymerase assay were submitted to LC-ESI⁺-MS/MS proteomic analysis (Fig. 5d; and Supplementary Fig. S34-35) which revealed three lysine residues participating in protein cross-linking to C^ESQ in newly synthesized RNA (K269 in sample 1; K462 and/or K463 in sample 2). We identified fragments corresponding to cross-linking to neighboring lysine residues in the spectrum of sample 2, but it can not be distinguished whether it is on K462 or K463. For sequence coverage of analysed protein samples see Supplementary Fig. S36-37 and Supplementary Table S4 in supporting material. The comparison with published crystal structure of JEV RdRp (PDB 4K6M) reveals the location of the K269 in 10-residue linker region (residues 266-275) which connects the MTase on its C-terminus (Supplementary Fig. S38). The sequence of this linker varies highly among genus flavivirus. The lysines K462 and K463 are in ring finger domain of RdRp (residues 453-479; Supplementary Fig. S38) that forms the roof of the NTP entry channel at the -5 position from the NTP binding motif F (Phe467). These amino acids are highly conserved in flaviviruses.⁵⁶

Herein we report synthesis of squaramate modified ribonucleotide C^ESQTP and its incorporation into the RNA by in vitro transcription. We have found that the spermidine (primary amine) present in the commercial reaction buffer interfered with the modification during transcription reaction. After optimization of reaction conditions by exchanging the primary amine for quaternary betaine, full-length RNA transcripts containing one to eight C^ESQ modifications were synthesized. We have also successfully used C^ESQTP in PEX synthesis of modified RNA with engineered TGK DNA polymerase in analogy to our recent publication.⁴¹ The ESQ-modified RNA was used in post-synthetic labelling with sulfo Cy-5-amine and conjugation with short Lys-containing peptide. The reactions proceeded with high conversion only in the presence of a large excess of the amine or peptide. Most importantly, the ESQ-modified RNA reacted with various lysine containing RNA-binding proteins (i.e., JEV and YFV NS5, and HIV-rt) to give the covalent cross-linked conjugates in conversions 9-43%. On the other hand, negative control experiments with non-binding or weakly binding proteins did not show any significant formation of covalent conjugates confirming that the proximity effect is crucial for good conversion. Finally, we investigated how modified triphosphate (C^ESQTP) affects the polymerase activity of RNA dependent RNA polymerases in vitro. Squaramate modification did not interfere with SARS-CoV-2 RdRp activity and resulted in full length RNA. On the other hand, experimental results show that newly synthesized RNA with incorporated C^ESQTP cross-links with JEV RdRp during polymerase reaction. Proteomic analysis of the reaction mixtures of JEV RdRp RNA extension identified lysine residues K269, K462 and K463 modified by C^ESQ from modified RNA. Hence there is a potential in using reactive modified nucleotides for inhibition of viral RNA polymerases, though probably not in the therapeutically relevant mode.

The squaramate moiety specifically reacts with primary amines including lysine to form stable amide bond under physiological conditions without any external reagent. Hence it has a good potential in post-synthetic labelling of RNA, bioconjugation of RNA with various biomolecules, cross-linking with proteins with abundant lysine or in developing irreversible inhibitors of RNA-binding enzymes containing free lysine residues (e.g., viral polymerases).

Full experimental part including the synthesis of the modified nucleoside (C^ESQTP), the corresponding NMR and MALDI data, detailed procedures on in vitro transcriptions, gel analysis of RNA constructs, other methods and notes can be found in the Supplementary Information. Main procedures are given below:

In vitro transcription with C^ESQTP

A solution of template DNA oligonucleotides (100 µM each) in 1:1 ratio in annealing buffer [Tris (10 mM, NaCl (50 mM), EDTA (1 mM), pH 7.8] was heated to 95°C for 5 minutes and slowly cooled to 25°C in 45 minutes. The resulting DNA (50 uM) was used as a template for transcription reactions. In vitro transcription reactions were performed in the total volume of 20 µL containing: dsDNA template (2 µM), ATP (1 mM), UTP (1 mM), GTP (0.8 mM), CTP (1 mM) for positive control, C^ESQTP (1.6 mM) for modified RNA, [α-³²P]-GTP (111 TBq/mmol, 370 MBq/mL, 0.2 µL), T7 RNA polymerase (2.5 U/µL), MgCl₂ (4.8 mM), dithiothreitol (DTT; 12 mM), Triton X-100 (0.12%), dimethyl sulfoxide (DMSO; 5%) and 1x transcription buffer B (40 mM Tris-HCl (pH 7.9), 6 mM MgCl₂, 10 mM DTT, 10 mM NaCl, 2mM betain). Water was used instead of the solution of C^ESQTP in the negative control experiment. The mixture was incubated at 37°C for
2 h

DNase I treatment

The DNA template was digested by DNase I to attain pure RNA. Transcription mixture (25 µL), 4 µL of DNase I buffer (supplied with the enzyme), 4 µL of DNAse I (4 U), and 7 µL DEPC water were incubated 60 min at 37°C. The enzyme was heat deactivated at 75°C for 10 min followed with cooling on ice. All samples were purified on NucAway Spin Columns (50 µL) for further use.

RNA Synthesis using TGK polymerase

Reactions were performed in a total volume of 10 µL. Reaction mixtures containing ssDNA template Prb4basII (4.8 µM final, Table S1), FAM-labelled DNA primer Prim248-short (4 µM final), three natural NTPs (0.8 mM final), C^ESQTP (0.8 mM final), TGK polymerase (1.5 µM final) and ThermoPol reaction buffer (1× final) were heated at 95°C for 30 sec and followed by 60°C for 2 h. Positive control was performed using natural CTP (0.8 mM final) and negative control in the absence of CTP/C^ESQTP. Reactions were then treated with TurboDNase (2U) and heated at 37°C for 30 min. Crude reaction mixture was purified using QIAquick (Qiagen) kit.

Cross-linking of ethoxy squarate‑modified RNA with sulfo-Cy-5-amine (35_RNA_1C^ESQ_Cy5)

Unlabelled natural RNA and RNA_1C^ESQ were prepared by in vitro transcription reaction described above. The reaction mixtures for sulfo-Cy-5-amine addition (10 µL) were prepared from purified transcribed product (5 μM) reconstituted in PBS buffer (pH 7.4) and sulfo-Cy-5-amine was added (2 mM). All samples were incubated on 37°C overnight. The products were purified on NucAway Spin Columns.

Cross-linking of ethoxy squarate‑modified RNA(35_RNA_1C^ESQ or 35_RNA_3C^ESQ) with peptide (Ac-Lys-Ala-Ala)

Reaction mixtures (40 µL total volume) containing 35_RNA_1C^ESQ or 35_RNA_3C^ESQ (2 µM final), tripeptide Ac-Lys-Ala-Ala (500/5000/9000 equiv.) and buffer (100 mM borate pH 9 or
20 mM carbonate-bicarbonate buffer pH 9.5) were heated at 37°C for 48 h. Crude reaction mixtures were purified using Monarch RNA cleanup kit

Cross-linking of 36_vRNA_1gC^ESQ with Bovine Serum Albumin (BSA) and Single Strand Binding Protein (SSB)

Radioactively labelled natural RNA and 36_vRNA_1gC^ESQ were prepared by in vitro transcription as described above. Natural or modified RNA (1µM) was incubated with 20 µM protein (BSA resp. SSB) in 10× binding buffer D (100 mM Tris pH. 8.0, 20 mM MgCl₂, 100 mM KCl, 12 mM ꞵME; 2µL), 50% glycerol (2 µL) and DEPC water (total reaction volume 20 µL) at 37°C. After 1 hour 5 µL of the reaction mixture was separated by 5-10% native PAGE (acrylamide/bisacrylamide 37.5:1; 4°C, 200V, 0.250 M Tris, 0.192 M glycine,). The rest of the reaction was incubated overnight at 37°C, then diluted with 2× VPS loading buffer, denatured 10 min at 95°C prior to loading and analysed by 5-10% SDS denaturing PAGE (acrylamide/bisacrylamide 37.5:1; 0.250 M Tris, 0.192 M glycine, 0.100% SDS) at room temperature (230 V, 70 min).

Cross-linking of 36_vRNA_1gC^ESQ, 36_vRNA_3fC^ESQ with Nonstructural Protein NS5 of Japanese Encephalitis Virus (JEV) and Yellow Fever Virus (YFV)

Radioactively labelled natural RNA and 36_vRNA_1gC^ESQ, 36_vRNA_3fC^ESQ (with one and three modifications respectively) were prepared by in vitro transcription as described above. Natural or modified RNA (0.500 µM) was incubated with 2 µM of NS5 protein (JEV resp. YFV) in 10× binding buffer E (50 mM Tris-HCl pH. 7.4, 100 mM DTT, 5% Triton X-100, 10% glycerol; 2µL), 10 mM MnCl₂ (2 µL), 10 mM MgCl₂ (2 µL), 50% glycerol (2 µL) and DEPC water (total reaction volume 20 µL) at 34°C. After 1 hour 3 µL of the reaction mixture was separated by 5-7% native PAGE (acrylamide/bisacrylamide 37.5:1; 4°C, 200 V, 1× Tris-glycine). The rest of the reaction was incubated 48 h at 34°C, then diluted with 2× VPS loading buffer, denatured 10 min at 95°C prior to loading and analysed by 5-10% SDS denaturing PAGE (acrylamide/bisacrylamide 37.5:1; 0.250 M Tris, 0.192 M glycine, 0.100% SDS) at room temperature (230 V, 70 min).

Cross-linking of 36_vRNA_1gC^ESQ, 36_vRNA_3fC^ESQ with SARS-CoV-2 RdRp

Radioactively labelled natural RNA and 36_vRNA_1gC^ESQ, 36_vRNA_3fC^ESQ (with one and three modifications respectively) were prepared by in vitro transcription as described above. Natural or modified RNA (1 µM) was incubated with 8 µM SARS-CoV-2 RdRp protein (complex of 2 µM SC nsp12 and 6 µM nsp7/8 proteins) in 10× binding buffer D (100 mM Tris-HCl pH. 8, 20 mM MgCl₂, 100 mM KCl 12 mM ꞵME; 2µL), 50% glycerol (2 µL) and DEPC water (total reaction volume 20 µL) at 30°C. After 1 hour 3 µL of the reaction mixture was separated by 10% native PAGE (acrylamide/bisacrylamide 37.5:1; 4°C, 200 V, 1× Tris-glycine). The rest of the reaction was incubated overnight at 30°C, then diluted with 2× VPS loading buffer, denatured 10 min at 95°C prior to loading and analysed by 5-10% SDS denaturing PAGE (acrylamide/bisacrylamide 37.5:1; 0.250 M Tris, 0.192 M glycine, 0.100% SDS) at room temperature (230 V, 70 min).

Cross-linking of 36_vRNA_1gC^ESQ, 36_vRNA_3fC^ESQ with SARS-CoV-2 nucleoprotein

Radioactively labelled natural RNA and 36_vRNA_1gC^ESQ, 36_vRNA_3fC^ESQ (with one and three modifications) were prepared by in vitro transcription as described above. Natural or modified RNA (1 µM) was incubated with 8 µM SARS-CoV-2 nucleoprotein in 10× binding buffer D (100 mM Tris-HCl pH. 8, 20 mM MgCl₂, 100 mM KCl 12 mM ꞵME; 2µL), 50% glycerol (2 µL) and DEPC water (total reaction volume 20 µL) at 30°C. After 1 hour 3 µL of the reaction mixture was separated by 10% native PAGE (acrylamide/bisacrylamide 37.5:1; 4°C, 200 V, 1× Tris-glycine). The rest of the reaction was incubated overnight at 30°C, then diluted with 2× VPS loading buffer, denatured 10 min at 95°C prior to loading and analysed by 5-10% SDS denaturing PAGE (acrylamide/bisacrylamide 37.5:1; 0.250 M Tris, 0.192 M glycine, 0.100% SDS) at room temperature (230 V, 70 min).

Cross-linking of 36_vRNA_1gC^ESQ with HIV-rt

Radioactively labelled natural RNA and 36_vRNA_1gC^ESQ (with one modification) was prepared by in vitro transcription as described above. Natural or modified RNA (0.500 µM) was incubated with 1 µM HIV-rt in buffer B (40 mM Tris-HCl (pH 7.9), 6 mM MgCl₂, 10 mM DTT, 10 mM NaCl, 2mM betain), 50% glycerol (2 µL) and DEPC water (total reaction volume 20 µL) at 37°C. After 1 hour 3 µL of the reaction mixture was separated by 10% native PAGE (acrylamide/bisacrylamide 37.5:1; 4°C, 200 V, 1× Tris-glycine). The rest of the reaction was incubated overnight at 37°C, then diluted with 2× VPS loading buffer, denatured 10 min at 95°C prior to loading and analysed by 5-10% SDS denaturing PAGE (acrylamide/bisacrylamide 37.5:1; 0.250 M Tris, 0.192 M glycine, 0.100% SDS) at room temperature (230 V, 70 min).

JEV and YFV RdRp mediated RNA extension assay⁵²

The polymerase activity of NS5 proteins was determined in a PEX reaction using fluorescently labelled RNA templates (Table S2). The reaction mixture (20 µl) contained reaction buffer (5 mM Tris-HCl pH. 7.4, 10 mM DTT, 0.100% Triton X-100, 1% glycerol, 1 mM MnCl₂, 1 mM MgCl₂), 10 μM NTPs, 200 nM template and 800 nM NS5 proteins. In the positive control, all natural NTPs were used and in the modified version 10 μM C^ESQTP instead of CTP was used. The reactions were incubated for 1 h and overnight respectively, at 34°C. Then the reactions were incubated with Proteinase K (1U) 30 min at room temperature. Reactions were stopped by adding of 2× PAGE stop solution and denatured at 95°C for 10 min prior to loading. Samples were separated by 12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under denaturing conditions (TBE 1×, 42 mA, 1 hour).

Immunodetection of 46_RNA_HP^C4_JEVNS5 conjugate

Natural 46_RNA_HP and 46_RNA_HP^C4were prepared by in vitro extension assay as described above. The reaction mixtures were diluted with 2× VPS loading buffer, denatured 10 min at 95°C prior to loading and electrophoresed on 5-10% SDS denaturing PAGE (acrylamide/bisacrylamide 37.5:1; 0.250 M Tris, 0.192 M glycine, 0.100% SDS) at room temperature (230 V, 70 min). The gel was blotted (dry; 0.250 M Tris, 0.192 M glycine, 10% methanol, 0.100% SDS; 12 V/2 hours at r.t.) to polyvinylidene fluoride Immobilon-P transfer membrane (Millipore). The membrane was washed with TBS (2.50 mM Tris, 15 mM NaCl, 0.200 mM KCl, pH 7.4), blocked with 5% non-fat milk (in T-TBS; 2.50 mM Tris, 15 mM NaCl, 0.200 mM KCl, 0.500% Tween-20, pH 7.4) for 30 min and afterwards washed with T-TBS (20 ml, 10 min). The membrane was incubated with JEV NS5 polyclonal antibody (rabbit/IgG; Invitrogen) at dilution 1:1000 in 5% non-fat milk in T-TBS at 4°C overnight. The unbound antibodies were washed out with T-TBS (20 ml, 3 × 5 min). Next, the membrane was incubated with anti-rabbit IgG HRP linked secondary antibody (Invitrogen) at 1:1000 dilution in 5% non-fat milk in T-TBS at 25°C for 1 hour. The access of antibodies was removed by washing with T-TBS (20 ml, 3 × 5 min) and TBS (20 ml, 10 min). The membrane was incubated with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) for 1 min.

SARS-CoV-2 RdRp mediated RNA extension assay⁵⁷

The polymerase activity of SARS-CoV-2 RdRp was determined in a PEX reaction using fluorescently labelled RNA templates (Table S2). The reaction mixture (20 µl) contained reaction buffer (10 mM Tris-HCl (pH. 8.0), 2 mM MgCl₂, 10 mM KCl, 1 mM ꞵME), 10 μM NTPs, 0.500 µM template, 1 μM nsp12 and 3 μM nsp7/8 proteins. In the positive control, all natural NTPs were used and in the modified version 10 μM C^ESQTP instead of CTP was used. The reactions were incubated for 1 h at 30°C. Reactions were stopped by adding of 2× PAGE stop solution and denatured at 95°C for 10 min prior to loading. Samples were separated by 12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under denaturing conditions (TBE 1×, 42 mA, 1 hour).

For MALDI TOF analysis, the reaction mixture was desalted with Bio-Spin6/Biorad columns (buffer was exchanged for water according to supplied protocol).

MALDI-TOF analysis of ethoxy squarate‑modified RNAs

Ethoxy squarate modified RNAs were prepared by In vitro transcription reactions described above. After DNase I treatment and spin-column purification, the products were freeze-dried, then redissolved in the water and analysed by MALDI-TOF mass spectrometry.

LC-MS characterization of FAM_31RNA_4C^ESQ, 35_RNA_1C^ESQ, 35_RNA_3C^ESQ, and their corresponding conjugates with peptide Ac-Lys-Ala-Ala (35_RNA_1C^ESQ-P 35_RNA_3C^ESQ-P)

Mass spectra of oligonucleotides and their conjugates with peptide (were measured on Agilent 1290 Infinity II Bio system with DAD detector and mass spectrometer MSD XT. LC-MS analyses were carried out using bioZen 1.7 µm Oligo, 2.1x150 mm column (Kinetex) using mobile phase A (15 mM Et₃N, 300 mM HFIP in H₂O) and mobile phase B (15 mM Et₃N, 300 mM HFIP in MeOH) by 30 min gradient from 5 % B to 50 % B. Deconvolutions of LC-MS spectra were carried out using UniDec program.⁵⁸

Intact mass measurement of 36_vRNA_1gC^ESQ_SSB

36_vRNA_1gC^ESQ containing one modification was cross-linked to SSB protein as described above. The volume of 18 μl of the sample was injected onto a MassPREP Micro Desalting column
(20 μm, 5-mm by 2.1-mm ID, Waters) and desalted and eluted by fast gradient (4 min). Mobile phase A (10 mM ammonium acetate in H₂O) and B (acetonitrile) were used for elution. The separation was carried out by AQUITY UPLC I-Class system (Waters) and was on-line coupled to Mass Spectrometer Synapt G2 (Waters) to acquire mass spectra using electrospray ionization in positive mode. The TOF mass range was set from m/z 500 to 4000. The raw spectrum was subtracted and deconvoluted (MaxEnt 1, Waters) to produce the final spectrum.

LC-MS analysis of digested 46_RNA_HP^C4_JEVNS5 samples after polymerase assay

The JEV NS5 mediated RNA extension assay was performed as described above (20 × 20 μl reaction mixture), except the reaction was incubated overnight. The reaction was followed by trypsine digestion of the protein to peptides and RNA digestion by RNase A/T1 mix.

Samples were dissolved in 15 μl of 0.1 TFA in H₂O and 3 μl of the sample was injected on an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to a Mass Spectrometer Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific). The peptides were trapped on a PepMap100 column (5 μm, 5 mm by 300-μm internal diameter (ID); Thermo Fisher Scientific) and desalted with 2% acetonitrile in 0.100% formic acid at a flow rate of 5 μL/min. Eluted peptides were separated using an EASY-Spray PepMap100 C18 analytical column (2 μm, 50-cm by 75-μm ID; Thermo Fisher Scientific). The 30-min elution gradient at a constant flow rate of 300 nL/min was set to start at 5% phase B (0.100% formic acid in 99.9% acetonitrile) and 95% phase A (0.100% formic acid). Then, the content of acetonitrile was increased gradually up to 50 % of phase B. The orbitrap mass range was set from m/z 350 to 2000 in the MS mode, and for ions with a charge state 2-6 the fragmentation spectra were acquired. A Proteome Discoverer 2.5 (Thermo Fisher Scientific) was used for identification of peptide and protein using Sequest HS and MS Amanda as search engines and databases of protein sequence and common contaminants.

Data Availability

Detailed procedures and data are given in Supplementary Information.

Acknowledgements

The work was funded by Czech Science Foundation (20-00885X to I. I. and M.H.) and by the Ministry of Education, Youth and Sports of the Czech Republic grant RNA for therapy (CZ.02.01.01/00/22_008/0004575 to M. O., T. S. Q. and M. H.).

Author contributions:

I.I. and M. H. designed the study, analyzed results and wrote the paper. I.I. performed the chemical synthesis, biochemistry, cross-linking experiments and RdRp assays. E.K. and E. B. produced the JEV, YFV and SARS-CoV-2 RdRps. R.P. measured and characterized NMR spectra. M.V. performed proteomic analysis. T.S.Q and M.O. performed the PEX and peptide cross-linking.

Competing interests:

The authors declare no competing interests

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

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