Synthesis of 5′-NAD-Capped RNA - ACS Publications - American

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Synthesis of 5’-NAD-capped RNA Katharina Höfer, Florian Abele, Jasmin Schlotthauer, and Andres Jäschke Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00072 • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 5, 2016

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Synthesis of 5’-NAD-capped RNA Katharina Höfer, Florian Abele, Jasmin Schlotthauer & Andres Jäschke. Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg, Germany. Correspondence should be addressed to AJ. ([email protected]). ABSTRACT: In prokaryotic organisms, certain regulatory RNAs have recently been found to be linked to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) at their 5’-ends. Biochemical and structural investigations of this new caplike RNA modification require synthetic access to pure NAD-RNA. Here we report a chemo-enzymatic approach to generate 5’NAD-capped RNA in high yields and purity under mild conditions. This approach uses unprotected 5’-monophosphate RNA synthesized either chemically or enzymatically, 5’,5’-pyrophosphate bond formation by phosphorimidazolide chemistry, and an enzymatic clean-up step. Thus, 5’-NAD-modified RNA can be synthesized independent of length, structure and nucleotide sequence. RNA is generally built from only four canonical nucleotides, but a multitude of chemical modifications decorate RNA molecules to support their diverse coding, structural, regula1 tory, and catalytic functions. Most known modifications occur at internal positions, while there is limited diversity at 2 the 5’-end. Generally, the 5’-end is a triphosphate, diphosphate, monophosphate, or hydroxyl, and recent evidence indicates that the 5’-status is an important determinant for 3-5 molecular recognition. In eukaryotes, the 5’-terminus of messenger RNAs is protected by a N7-methylated guanosine cap, which is connected to the RNA via a 5’,5’-triphosphate 6-8 linkage. In 2009, the enzymatic cofactor nicotinamide adenine dinucleotide (NAD) has been reported as a covalent 9 modification of prokaryotic RNA. However, using a LC-MS based technique that requires complete digestion of RNAs to identify the NAD cap, the sequences that bear this modification had initially remained unknown. Recently, our group reported the development of a NAD-specific chemoenzymatic capture approach which allowed us to specifically enrich NAD-RNA from Escherichia coli total RNA and to 10 analyze it by next-generation sequencing. Among the captured RNAs, regulatory RNAs were found to be particularly abundant and were further confirmed by different biochemical analyses, including enzymatic digests and mass spectrometry. We have further discovered that the NADmodification stabilizes RNA against degrading enzymes, in a way similar to a eukaryotic cap. Another enzyme (NudC) 10 was found to decap NAD-modified RNA. This first description of capped RNA species in prokaryotes, and of a prokaryotic decapping machinery opens up a new field in prokary11 otic RNA biology. Given the central role of NAD in redox biochemistry, post-transcriptional protein modification, and signalling, its attachment to RNA points to unknown roles of RNA in these processes and to undiscovered pathways in 11-13 RNA metabolism and regulation in prokaryotes. To unravel these roles, the biosynthesis and degradation of such RNAs and the regulation of the underlying processes, robust methods for the preparation of large amounts of pure, homogenous 5’-NAD-RNA are required. Ideally, such methods should allow the preparation of RNAs of any size, se-

quence, and structure. The only published method uses in vitro transcription by T7 RNA polymerase, using high concentrations of NAD as “initiator nucleotide” that is selectively incorporated at the 5’-end. This method is, however, restricted to certain sequences (always starting with an adenosine), very inefficient for short transcripts, and inevitably 14-15 produces a mixture of NAD- and triphosphate-RNA. Methods to chemically synthesize such NAD-capped RNAs have not been reported so far. Direct incorporation during solid-phase synthesis shows little promise due to the lability 16 of nicotinamide riboside. However, there is a large body of 17-28 literature on the synthesis of NAD analogs. The challenge was finding a method that ideally allows site-specific functionalization of unprotected RNA with its multitude of functional groups. There are four conceivable ways to attach a nicotinamide riboside to an existing RNA molecule (Figure 1). We decided to focus on approach (1), namely on the reaction of a 5’monophosphate-RNA with activated nicotinamide mononucleotide (NMN). On the nucleotide level, the utilization of carbonyldiimidazole (CDI) for 5’-phosphate activation and subsequent pyrophosphate formation has been reported to 29-30 be mild, robust, and high-yielding. Such phosphorimidazolides are known to react with numerous nucleophiles, such as pyrophosphates or nucleoside mono-, di-, or tri30-34 phosphates. Numerous NAD analogs have been prepared 18-19, 26-27 by phosphorimidazolide chemistry. On the oligonucleotide level, imidazolide activation has been used exten35-36 37 sively for conjugation, non-enzymatic ligation, and 38 polymerization reactions, as well as for the preparation of 39-40 adenylylated and capped RNAs. These reports indicate that side reactions of the phosphorimidazolide with undesired nucleophiles (e.g., exocyclic amines of nucleobases) are not a major problem. NMN-phosphorimidazolide (Im-NMN) was synthesized as 29 41 described with some modifications. Starting from NMN, Im-NMN-2’-3’-carbonate was prepared using a 10x excess of CDI in dry DMF. Hydrolysis of this intermediate with 0.2 M TEAB furnished Im-NMN in 63% yield and high purity (Figure S1 and S2).

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Figure 1. Possibilities to attach nicotinamide riboside (N) to a RNA molecule and to synthesize 5’-NAD-RNA. Application of nucleophilic substitution reactions for the formation of 5’,5’-pyrophosphate bonds. (LG = Leaving group, N = Nicotinamide riboside, p = phosphate) As proof of principle, NAD was synthesized by reaction of Im-NMN and adenosine monophosphate (AMP) in DMF supplemented with MgCl2. HPLC analysis revealed 80% conversion and very little side products (Figure S3), encouraging us to transfer the method from AMP to 5’-phosphate-RNA.

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kinds of in vitro transcription, dephosphorylation of triphosphate RNA, or ribozyme cleavage of precursor sequences. For our investigation, we used a chemically 5’phosphorylated 20mer prepared by solid-phase synthesis and a 38mer prepared by enzymatic removal of pyrophosphate from an in vitro transcribed 5’-triphosphate RNA (Figure 2A and S5). 5’-P-RNA prepared by both methods was treated with a 1000x excess of Im-NMN in aqueous solution (50 mM MgCl2, 50°C, 2h) (Figure 2B). Cap formation was monitored by mass spectrometry (Figure 3 and S4). Purity and yields were analyzed by denaturing polyacrylamide electrophoresis (Figure 4 and S5). These analyses revealed the formation of new compounds, yielding mixtures of 5’-NAD-RNA and unreacted 5’-P-RNA.

Figure 3: Monitoring of NAD-RNA synthesis. MALDI mass spectra of 5’-P-RNA 20mer before (A) and after (B) imidazolide reaction, and after Xrn-1 treatment (C). While for short RNAs, a preparative chromatographic or electrophoretic separation of 5’-NAD-RNA and 5’-P-RNA appears feasible, it becomes increasingly difficult and inefficient with increasing molecular weight. We therefore considered the use of exonucleases that are specific for a mono42-44 phosphorylated 5’-terminus, such as Xrn-1 from yeast. (Figure 2B). While complete digestion of 5’-P-RNA was observed by denaturing PAGE (Figure 4 and S5), NAD-RNA was not attacked by the enzyme (Figure S6). Xrn-1 treatment of a mixture of 5’-NAD- and 5’-P-RNA obtained after imidazolide coupling resulted in depletion of 5’-P-RNA (Figure 3C and 4). NAD-RNA could then be easily purified by phenol/chloroform extraction, followed by ethanol precipitation. Mass spectrometry (MALDI and HR-ESI, LC-MS studies) confirmed the correct composition of the synthesized NAD-RNA (Figure 3C and S7). NAD-capped RNA was synthesized with yields of ~45 %. Figure 2. Synthesis of 5’-NAD-capped RNA. (A) Digestion of enzymatically synthesized 5’-triphosphate-RNA with commercially available polyphosphatase. (B) Im-NMN is coupled to the 5’-monophosphate group of the RNA. Remaining unreacted 5’-P-RNA is removed by exonuclease (Xrn-1, commercially available) treatment. There are many methods known to synthesize 5’-P-RNA of various lengths, from direct solid-phase synthesis to enzymatic phosphorylation with polynucleotide kinase to various

In addition to installing a nicotinamide adenine dinucleotide (NAD) at the 5’-end of RNA, the phosphorimidazolide coupling also allowed preparation of the nicotinamide guanosine (NGD), uridine (NUD) and cytidine (NCD) dinucleotide RNA analogs which are required for biochemical investigations and cannot be synthesized by any other known means. Isolated yields were between 30 and 45% (Figure 5A, S8A and S8B).

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Bioconjugate Chemistry to produce NAD-modified RNA in scales up to 20 nmol. The method will facilitate the elucidation of the biological relevance of NAD capping in pro- and eukaryotes, the identification of binding partners, players and mechanisms in biosynthesis and turnover. Furthermore, we expect the expansion of this chemistry to other types of cofactor-modified RNAs.

Figure 4: Treatment of 5’-P-RNA and 5’-NAD-RNA (20mer) with 5’-P dependent exonuclease Xrn-1. Analysis of complete digest of 5’-P-RNA by denaturing gel electrophoresis. To further support the identity of the chemo-enzymatically synthesized NAD-RNAs, we subjected them to decapping by the Nudix hydrolase NudC, which was overexpressed and 10 purified as described by Cahová et al.. We had previously discovered that this enzyme is able to hydrolyze the pyrophosphate bond both in enzymatically synthesized NADRNA, and in NAD-modified RNA isolated from E. coli, and therefore constitutes the first prokaryotic decapping enzyme. To analyze the acceptance as substrate of our synthetic 5’-NAD-RNA by NudC, we phosphorylated in vitro tran32 scribed RNA using polynucleotide kinase and γ-[ P]-ATP, 32 thereby installing a [ P]-monophosphate at the 5’-terminus. Radioactively labeled 5’-P-RNA was converted into 5’-NADRNA by imidazolide reaction, purified by Xrn-1 digestion as described above, and subjected to NudC hydrolysis in the presence of alkaline phosphatase. Alkaline phosphatase alone was unable to remove the radioactive label, consistent with the assumption of a “protected” (i. e., internal) radioactive phosphate. In the presence of NudC and alkaline phosphatase, however, the label was rapidly removed (Figure 5B and S8C), demonstrating that NMN moieties are indeed connected to the RNA via a 5’,5’-pyrophosphate bond. The rate of NAD cleavage was comparable to that measured for 10 NAD-RNA prepared by transcription initiation with NAD.

Figure 5. (A) Chemo-enzymatic synthesis of 5’-NXD-RNA. Shown are coupling efficiencies of NMN to 5’-P-RNA depending on the first nucleotide (A, G, C, U). (B) Enzyme kinetics of NudC on NAD-capped RNA (NppA-RNA). 32 Decapping was studied on [ P]-labelled NppA-RNA (position of the radiolabel marked with red asterisk) in the presence of an excess of alkaline phosphatase (AP), NudC or its inactive mutant NudC E178Q and analyzed by denaturing gel electrophoresis. As a biological relevant target, 5’-NAD capped RNA I (106 nt) was used. The approach described here allows the preparation of pure 5’-NAD-RNA in quantities sufficient for biochemical, mechanistic, and crystallographic investigations. It is likely independent of length and nucleotide sequence and also compatible with the introduction of further modified nucleotides. Using our chemo-enzymatic synthesis protocol, we are able

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental procedures, characterization data, and supplementary figures (PDF)

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the German Research council (DFG SPP 1784), grant # Ja 794/10-1. We thank Heiko Rudy for mass spectrometry analysis, Gabriele Nübel for LC-MS support and Fiona Berger for preparation of in vitro transcription reactions. REFERENCES (1) Helm, M., and Alfonzo, J. D. (2014) Posttranscriptional RNA Modifications: Playing Metabolic Games in a Cell's Chemical Legoland. Chem. Biol. 21, 174-85. (2) Machnicka, M. A., Milanowska, K., Oglou, O. O., Purta, E., Kurkowska, M., Olchowik, A., Januszewski, W., Kalinowski, S., Dunin-Horkawicz, S., Rother, K. M., et al. (2013) MODOMICS: a database of RNA modification pathways-2013 update. Nucleic Acids Res. 41, D262-D67. (3) Hornung, V., Ellegast, J., Kim, S., Brzozka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K. K., Schlee, M., et al. (2006) 5'-Triphosphate RNA is the ligand for RIG-I. Science 314, 994-7. (4) Goubau, D., Schlee, M., Deddouche, S., Pruijssers, A. J., Zillinger, T., Goldeck, M., Schuberth, C., Van der Veen, A. G., Fujimura, T., Rehwinkel, J., et al. (2014) Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates. Nature 514, 372-5. (5) Mackie, G. A. (1998) Ribonuclease E is a 5'-end-dependent endonuclease. Nature 395, 720-3. (6) Topisirovic, I., Svitkin, Y. V., Sonenberg, N., and Shatkin, A. J. (2011) Cap and cap-binding proteins in the control of gene expression. Wiley Interdisc. Rev.-RNA 2, 277-98. (7) Shatkin, A. J., and Manley, J. L. (2000) The ends of the affair: capping and polyadenylation. Nat. Struct. Biol. 7, 838-42. (8) Shatkin, A. J. (1976) Capping of eucaryotic mRNAs. Cell 9, 645-53. (9) Chen, Y. G., Kowtoniuk, W. E., Agarwal, I., Shen, Y., and Liu, D. R. (2009) LC/MS analysis of cellular RNA reveals NADlinked RNA. Nat. Chem. Biol. 5, 879-81.

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