Catalysts for RNA and DNA modification

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ScienceDirect Catalysts for RNA and DNA modification Dennis Gillingham1 and Ramla Shahid2 To study DNAs and RNAs it is often necessary to chemically modify them. Nature’s strategy for nucleic acid modification is to use selective catalysts, and chemists have begun to emulate this conceptual approach. In this review we present a summary of catalytic approaches toward the construction of modified RNAs and DNAs and outline our opinions on where new research is needed. Addresses 1 St. Johanns-Ring 19, Basel 4056, Switzerland 2 COMSATS Institute of Technology, Park Road, Islamabad, Pakistan Corresponding author: Gillingham, Dennis ([email protected])

Current Opinion in Chemical Biology 2015, 25:110–114 This review comes from a themed issue on Biocatalysis and biotransformation Edited by Thomas R Ward

http://dx.doi.org/10.1016/j.cbpa.2014.12.025 1367-5931/# 2015 Elsevier Ltd. All rights reserved.

Introduction Prokaryotes employ an RNA-guided endonuclease in their immune systems. The discovery and reengineering of this CRISPR/Cas9 system is revolutionizing biological research and may impact human health [1]. How could such a fundamental aspect of prokaryote biology remain unknown until 2007? Because what we can study is limited by what we can see. The need for ever-more sensitive and selective ways to visualize nucleic acids is the starting point for the present review. One of the great contributions of synthetic chemistry to the scientific enterprise is the solid-phase synthesis of oligonucleotides. Through this iterative approach access to any imaginable oligonucleotide is possible, but often difficult. The inherently linear strategy of solid-phase synthesis imposes a size limitation, and non-canonical phosphoramidite building blocks are challenging to synthesize, particularly for RNA. If larger constructs bearing sequence-defined internal alterations are needed fragments derived from polymerase synthesis are combined with synthetic fragments through splint-mediated ligation, a tedious and sometimes unpredictable process. Nevertheless, the study of DNA and RNA is greatly Current Opinion in Chemical Biology 2015, 25:110–114

facilitated by access to chemically modified derivatives. In fact even unselective modifications introduced by promiscuous activity of natural polymerases can be used in vivo to study transcriptomics [2] and genomics [3]. Modifications typically confer some observable property that reports on the production, folding, or binding interactions of nucleic acids. Modifications can also be used to alter the biological role of a given nucleic acid. While most modified nucleic acids are prepared by solid phase synthesis, direct modification would be more efficient; but such a strategy is faced with the challenge of the enormous functional group redundancy of large nucleic acids. Nature uses selective catalysts to achieve selective nucleic acid modification and here we will explore how researchers are building a toolkit of catalysts that emulate and expand on Nature’s approach.

Retooling natural enzymes Studying or reprogramming biopolymers at the molecular level often requires precise labeling. Chemical modification or synthetic labeling of RNA is expensive, time consuming, and at present only possible with short stretches of the polymer. In addition, labeling large RNAs continues to be a great challenge, but the emerging recognition of the importance of long non-coding RNAs provides the impetus for continued development. Nature has evolved many enzymes that interact with DNA or RNA specifically, and expanding on Nature’s designs might offer a mild and selective approach for modifying DNA or RNA. Natural proteins identify their target DNA or RNA sequence and act either by simply binding or modifying it covalently. DNA Methyltransferases (MTases) are one such group of enzymes, these bind a specific sequence in DNA or RNA and label it with a methyl group. MTases catalyze the nucleophilic attack of Cytosine (C), Adenine (A), or guanine (G) on to the electrophilic methyl group of its cofactor S-Adenosyl-Lmethionine (Adomet). Although MTases are wonderfully specific, the methyl group is a poor reporter, unless radioactively labeled with 14C or tritium, and therefore a number of researchers have examined the tolerance of MTases to Adomet analogues. In pioneering work, DNA Mtase from Thermus aquaticus (Taq) was used to fluorescently label duplex DNA [4]. A modified cofactor bearing a fluorescent dansyl group at C8 of adenine and an aziridine in the 50 -position did not interrupt binding to the enzyme or the target DNA, allowing MTase to couple the modified cofactor to its normal TCGA recognition site in the duplex. This sequence specific methyltransferase induced labeling opened the door to site-specific labeling of DNA with a range of reporter groups. Furthermore, the great variety of MTases with unique binding sites www.sciencedirect.com

Catalysts for RNA and DNA modification Gillingham and Shahid 111

suggests a broadly applicable technology. A shortcoming of the method, however, is that the entire cofactor is integrated in the target DNA in a nitrogen mustard-type reaction, a rather drastic change that might not be tolerated in functional RNAs. The same group, in collaboration with the Klimasauskas group, therefore developed Adomet analogues that bear alkyl groups in place of methyl (see Panel a, Figure 1 for examples). Linear alkyl, alkenyl, and alkynyl were all effective cofactors (branched alkyls were unreactive), with the unsaturated derivatives being most active, delivering up to 32 turnovers per hour [5]. The approach has been extended to RNA MTases that act on tRNAs along with Adomet substrates that are amenable to further derivatization reactions such as the copper-catalyzed azide-alkyne cycloaddition [6]. Recently the RNA MTase HEN1 from Arabidopsis thaliana has been used to achieve the site-specific labeling of small

duplex RNAs, such as those found in the microRNA and siRNA pathways [7]. An exciting development for RNA labeling is the successful coopting of a C/D box ribonucleoprotein (RNP) MTase from the thermophilic archaeon Pyrococcus abyssi [8]. This RNP MTase uses a twelve nucleotide RNA guide sequence to precisely base pair to its target and methylates the 20 OH of the nucleotide five base-pairs upstream of the D box. The method has dual advantages over previous efforts: the enzyme can be reprogrammed by simply modifying the RNA guiding sequence in the RNP and the best performing Adomet analogue was the propargyl variant, facilitating further couplings with any desired azide through the CuAAC. Further work is required, however, since the transfer efficiency was only 5–10%.

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(a) S-adenosylmethionine (Adomet) is the main source of electrophilic methyl in Nature, Adomet analogues (see boxed structures) are also tolerated by many methyltransferases (MTases); (b) Various DNA and RNA sequences and structural motifs have been targeted with Adomet analogues; (c) The 50 -cap structure of eukaryotic mRNAs can be targeted with Adomet analogues through the action of trimethylguanosine synthases (Tgs). www.sciencedirect.com

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The Rentmeister group has developed methods to use human trimethylguanosine synthases, which convert standard m7G caps in eukaryotic mRNA to the 2,2,7trimethylguanosine, or trimethylguanosine synthases from Giardia lamblia (GlaTgs), which normally add a single methyl group to N2 position of m7GDP, m7GTP, and m7GpppA using Adomet or its analogues. The enzyme’s ability to accommodate modified analogues was then further exploited to add unsaturated functional groups, such as terminal alkynes, to the 50 -cap structure of mRNAs in vitro and in cell lysates (see Panel c, Figure 1). These could be subsequently fluorescently labeled using CuAAC. Homology modelling guided the introduction of a point mutation to create a variant, GlaTgs-Var1, which was better expressed and more efficient (fivefold increase in kcat/Km) for the unnatural Adomets [9]. The use of CuAAC is toxic to cells due to the redox damage imposed by Cu(I). A variation of the mRNA labeling method was therefore developed that uses the strain promoted azide–alkyne cycloaddition (SPAAC) for conjugation [10]. The mRNA labeling strategy could offer the means to explore mRNA localization, dynamics, and degradation without using complicated hybridization probes or engineered tags. At its present stage of development the mRNAs would have to be introduced by transfection, but by employing recently developed strategies for creating oligonucleotide guided Tgs variants [11], future efforts may make this technology amenable to selective in vivo labeling.

Ribozymes and deoxyribozymes Although DNA and RNA have less functional group diversity than proteins, their programmable and evolvable binding properties have spurred the development of DNA and RNA-based catalysts (often termed DNAzymes for DNA and ribozymes for RNA). The Silverman lab has been a pioneer in developing DNAzymes for cleavage, ligation, or modification reactions of nucleic acids. In 2007 they reported a deoxyribozyme-catalyzed RNA labeling method that resembles certain aspects of natural RNA splicing [12]. In their system a ‘tagging RNA’ is coupled to the 20 -hydroxyl of an adenosine on a target RNA in a site-specific reaction. The tagging RNA bears an aminoallylcytosine that is pre-labeled with any type of biophysical probe through amide formation with activated esters. It also bears a 50 -triphosphate, making it primed for reactions with hydroxyl groups. Through specific 3D-folding and Watson–Crick base-pairing the DNAzyme then holds the target RNA in proximity to the 50 -triphosphate of the tagging RNA. The 20 -hydroxyl of an internal adenosine then attacks the 50 -triphosphate to create a branched RNA bearing the reporter group. This work was groundbreaking because it demonstrated sitespecific internal modification of a long native RNA. Nevertheless the strategy has not been widely adopted, likely because it is complex to orchestrate and it forces the integration of an RNA fragment (8–17 nucleotides) Current Opinion in Chemical Biology 2015, 25:110–114

at the tagging site, which must be tolerated in the target application. A recent report from the Ho¨bartner lab has eliminated many of the shortcomings of the first generation RNA tagging DNAzymes [13]. Building from earlier work [14], they have shown that DNAzymes can catalyze the coupling of various guanosine mononucleotide triphosphates to the 20 -hydroxyl of an internal adenosine of a target strand. The ability to use a mononucleotide eliminates the problem of having a leftover oligoribonucleotide at the branch site. These authors also discovered that terbium(III) can substantially accelerate the rate of the ligation reaction. Using terbium-assisted DNAzymes, they could achieve multiple labeling of large complex RNAs with a variety of biophysical reporters in high yields. Perhaps the most important feature of the Ho¨bartner approach is the ease of implemention: aside from the target RNAs all components can be purchased. This technique has great potential for creating large site-specifically modified RNAs for research. Imaging of RNAs in their native environment is a difficult and largely unsolved problem [15,16]. As groundbreaking discoveries relating to the transcriptome unfold each year (e.g., small interfering RNAs, microRNAs, long noncoding RNAs, CRISPR RNAs), methods for the selective in vivo visualization of RNAs become increasingly urgent. Furthermore, extensive cross-talk between the different components of the transcriptome [17] imply that the function of a specific RNA can only be understood in the context of the whole system; in other words in vivo studies are essential for understanding RNAs. One strategy is to engineer RNA motifs that bind reporter molecules into larger RNAs. A green fluorescent protein (GFP) fusion with the phage coat protein MS2 is the most widely used approach. It relies on the high affinity and selectivity of the MS2 protein for a specific hairpin RNA sequence that normally resides in the phage genome. Although MS2 tagging is powerful, the high background fluorescence, and the uncertainty of whether engineered changes to the RNA alter its biology fuel the demand for alternatives. Engineered aptamers that bind fluorescent or fluorogenic small molecules have recently been developed and applied to in vivo RNA detection [18,19]. Since fluorescence is only turned on upon aptamer binding these techniques have less background than MS2 detection; they suffer, however, from a lower binding affinity. All of the current techniques apply non-covalent interactions and therefore rely on slow koff for efficient detection. Recently self-alkylating ribozymes have been evolved that covalently link a fluorescein derivative to RNA [20]. Although iodoacetamide is a powerful electrophile that can alkylate nucleobases, the reaction is normally slow. Using systematic evolution of ligands by exponential enrichment (SELEX), Sharma and coworkers evolved ribozymes that dramatically accelerate the alkylation www.sciencedirect.com

Catalysts for RNA and DNA modification Gillingham and Shahid 113

process (k2 = 100 M1 s1, see Panel c in Figure 2). Although the technique has not yet been used in vivo, the covalent link could offer unique advantages such as the ability to imbue the RNA with cross-linking groups to capture protein partners, or affinity tags for pull-down assays. Furthermore the power of the SELEX technique should facilitate the evolution of new ribozymes with specificity for different small molecules, providing a strategy for the simultaneous visualization of multiple RNAs.

small oligonucleotides the efficiency and reliability of solid-phase synthesis is hard to beat. However, large RNAs such as long non-coding RNAs or mRNAs have exciting potential as gene therapy drugs [21] or vaccine antigens [22], and for such molecules post-transcriptional chemical modification will be crucial. My group has been developing organometallic catalysts for post-synthetic modification of DNA and RNA. We have found that donor–acceptor substituted carbenes stabilized by Rh(II) dimers [23] or Cu(I) [24] selectively target the exocyclic amine nitrogens of nucleobases (N4C, N2G, and N6A). Although not sequence specific, the reaction is structure specific, targeting bases in single-stranded regions of large folded nucleic acids. Although we are still at the discovery phase of this technology, already in its present unselective form it has been used for fluorescent labeling in PCR reactions, and could be used for chemical tailoring of aptamers or in the SHAPE analysis of RNAs.

Organometallic catalysis The emergence of nucleic acid derived or inspired medicines will demand new chemical methods for tailoring the distribution, metabolism, and pharmacokinetic properties of these macromolecules. While enzymatic techniques may play a role, for industrial production chemical methods will likely prove more economical. Furthermore, purely artificial catalysts could offer reaction manifolds inaccessible to enzymes, ribozymes, or DNAzymes. For Figure 2

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(a) A DNAzyme catalyses the transfer of an 8–17 nucleotide tagging RNA bearing a reporter tag; (b) A DNAzyme can couple a guanine mononucleotide bearing a reporter tag; (c) A self-ligating ribozyme identified through SELEX accelerates nucleobase alkylation with fluorescein iodoacetamide. www.sciencedirect.com

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Conclusions Advances in sequencing and other analytical techniques have revealed that RNA functions are extraordinarily complex and interconnected. Just as solid-phase synthesis was a crucial component of the molecular biology revolution, the preparation of large modified RNAs will be critical for parsing the transcriptome and for creating RNAs with new functions. We have presented here a snapshot of the catalytic strategies toward direct RNA modification. The picture that emerges shows that in vitro modification is becoming more powerful and many forms of RNA can be labeled in a site-specific way with catalysts; these techniques will be a great boon for in vitro biophysics studies. A transformative innovation would be the ability to label and track individual transcripts in vivo with small molecule reporter tags. The next decade will surely witness exciting developments in this direction.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. Pennisi E: The CRISPR craze. Science 2013, 341:833-836.  An overview of the recent discovery and reengineering of the CRISPR/Cas system. 2.

Jao CY, Salic A: Exploring RNA transcription and turnover in vivo by using click chemistry. Proc Natl Acad Sci USA 2008, 105:15779-15784.

3.

Rieder U, Luedtke NW: Alkene–tetrazine ligation for imaging cellular DNA. Angew Chem Int Ed 2014, 53:9168-9172.

4. 

Pljevaljcic G, Pignot M, Weinhold E: Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA. J Am Chem Soc 2003, 125:3486-3492. The first demonstration that a methyltransferase could be coopted for DNA labelling.

Dalhoff C, Lukinavicius G, Klimasa˘uskas S, Weinhold E: Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases. Nat Chem Biol 2006, 2:31-32. Proof-of-concept that alkyl groups larger than methyl are tolerated by methyltransferases.

5. 

6.

Motorin Y, Burhenne J, Teimer R, Koynov K, Willnow S, Weinhold E, Helm M: Expanding the chemical scope of RNA: methyltransferases to site-specific alkynylation of RNA for click labeling. Nucleic Acids Res 2011, 39:1943-1952.

Plotnikova A, Osipenko A, Masevicˇius V, Vilkaitis G, Klimasˇauskas S: Selective covalent labeling of miRNA and siRNA duplexes using HEN1 methyltransferase. J Am Chem Soc 2014, 136:13550-13553. Establishes the possibility of targeting short duplex RNAs with methyltransferases.

7. 

8. 

Tomkuviene M, Clouet-d’Orval B, Cerniauskas I, Weinhold E, Klimasauskas S: Programmable sequence-specific clicklabeling of RNA using archaeal box C/D RNP methyltransferases. Nucleic Acids Res 2012, 40:6765-6773. First example of an RNA-guided methyltransferase to control siteselectivity.

Current Opinion in Chemical Biology 2015, 25:110–114

9.

Schulz D, Holstein JM, Rentmeister A: A chemo-enzymatic approach for site-specific modification of the RNA cap. Angew Chem Int Ed Engl 2013, 52:7874-7878.

10. Holstein JM, Schulz D, Rentmeister A: Bioorthogonal sitespecific labeling of the 50 -cap structure in eukaryotic mRNAs. Chem Commun (Camb) 2014, 50:4478-4481. 11. Vogel P, Schneider MF, Wettengel J, Stafforst T: Improving site directed RNA editing in vitro and in cell culture by chemical modification of the guide RNA. Angew Chem Int Ed 2014, 53:6267-6271. Establishes a general approach for creating RNA-guided catalysts in cells. 12. Baum DA, Silverman SK: Deoxyribozyme-catalyzed labeling of  RNA. Angew Chem Int Ed 2007, 46:3502-3504. Proof-of-concept that DNAzymes can label RNA. 13. Bu¨ttner L, Javadi-Zarnaghi F, Ho¨bartner C: Site-specific labeling of RNA at internal ribose hydroxyl groups: terbiumassisted deoxyribozymes at work. J Am Chem Soc 2014, 136:8131-8137. 14. Ho¨bartner C, Silverman SK: Engineering a selective smallmolecule substrate binding site into a deoxyribozyme. Angew Chem 2007, 119:7564-7568. 15. Armitage BA: Imaging of RNA in live cells. Curr Opin Chem Biol 2011, 15:806-812. 16. Santangelo PJ, Alonas E, Jung J, Lifland AW, Zurla C: Probes for intracellular RNA imaging in live cells. In Methods in enzymology, vol 505. Edited by Conn . Academic Press; 2012:383399 (Chapter 20), http://www.sciencedirect.com/science/article/ pii/B9780123884480000280. 17. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi Pier P: A ceRNA  hypothesis: the rosetta stone of a hidden RNA language? Cell 2011, 146:353-358. Introduces the concept that various RNA forms communicate in an intricate network based on Watson–Crick interactions. 18. Paige JS, Wu KY, Jaffrey SR: RNA mimics of green fluorescent  protein. Science 2011, 333:642-646. Proof-of-concept for aptamer labelling in conjunction with small molecule turn-on fluorophores. 19. Dolgosheina EV, Jeng SCY, Panchapakesan SSS, Cojocaru R, Chen PSK, Wilson PD, Hawkins N, Wiggins PA, Unrau PJ: RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem Biol 2014, 9: 2412-2420. 20. Sharma AK, Plant JJ, Rangel AE, Meek KN, Anamisis AJ, Hollien J, Heemstra JM: Fluorescent RNA labeling using self-alkylating ribozymes. ACS Chem Biol 2014, 9:1680-1684. 21. Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Spater D, Xu H, Tabebordbar M, Gorbatov R, Sena B et al.: Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat Biotech 2013, 31:898-907. 22. Kallen K-J, Theß A: A development, that may evolve into a revolution in medicine: mRNA as the basis for novel, nucleotide-based vaccines and drugs. Ther Adv Vaccines 2013, 2:10-31. 23. Tishinov K, Schmidt K, Ha¨ussinger D, Gillingham DG: Structure selective catalytic alkylation of DNA and RNA. Angew Chem Int Ed 2012, 51:12000-12004. First demonstration of organometallic catalysis for nucleic acid labelling. 24. Tishinov K, Fei N, Gillingham D: Cu(I)-catalysed N-H insertion in water: a new tool for chemical biology. Chem Sci 2013, 4: 4401-4406.

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