Applications of Phosphate Modification and Labeling to Study (m)RNA Caps
- 1.4k Downloads
The cap is a natural modification present at the 5′ ends of eukaryotic messenger RNA (mRNA), which because of its unique structural features, mediates essential biological functions during the process of gene expression. The core structural feature of the mRNA cap is an N7-methylguanosine moiety linked by a 5′–5′ triphosphate chain to the first transcribed nucleotide. Interestingly, other RNA 5′ end modifications structurally and functionally resembling the m7G cap have been discovered in different RNA types and in different organisms. All these structures contain the ‘inverted’ 5′–5′ oligophosphate bridge, which is necessary for interaction with specific proteins and also serves as a cleavage site for phosphohydrolases regulating RNA turnover. Therefore, cap analogs containing oligophosphate chain modifications or carrying spectroscopic labels attached to phosphate moieties serve as attractive molecular tools for studies on RNA metabolism and modification of natural RNA properties. Here, we review chemical, enzymatic, and chemoenzymatic approaches that enable preparation of modified cap structures and RNAs carrying such structures, with emphasis on phosphate-modified mRNA cap analogs and their potential applications.
KeywordsRNA labeling Capping Molecular probe Nucleotide Cap analog 7-methylguanosine
1 RNA Cap Structures and Their Functions
1.1 7-Methylguanosine mRNA Cap
Synthetic cap analogs are useful research tools that facilitate investigation of cap-related processes both at a molecular level and in biochemical and biological experiments, which may eventually lead even to development of novel therapeutic approaches . In this paper, we review approaches employed to develop cap-based molecular tools that could benefit biological studies, with the focus on phosphate-labeled and phosphate-modified cap analogs of m7G cap and other structurally related RNA cap structures.
1.2 m7G Cap 0, 1, 2 and Other Cap Structures
Although the term cap usually refers to the m7G moiety linked to the 5′ end of a transcript, different versions of the m7G cap have been recognized along with other types of cap structures found on different RNA types and in various organisms. Depending on the methylation pattern of the first few 5′-nucleotides in an m7G-capped RNA body, cap variants referred to as cap 0, cap 1, cap 2, and so on, can be distinguished (Fig. 1b). In addition, other types of methylated cap structures have been identified in eukaryotes, trimethylguanosine and γ-methyl cap being the prime examples (Fig. 1c). Finally, it was recently discovered that prokaryotes also synthesize conjugates of RNA with small molecules that structurally resemble eukaryotic 5′ caps (Fig. 1d).
Cap 0 is m7G cap without additional methylations in the RNA body. However, cap 0 can undergo further methylations at the 2′-O position within the ribose of the first or within the first and second nucleotide to produce cap 1 or cap 2, respectively (Fig. 1b) . Generally, cap 0 structures are more common in lower eukaryotes, whereas cap 1 and cap 2 structures are found in higher organisms, including mammals . Additional methylations at the first and second nucleobase of mRNAs can also take place in cap 1 and 2. For a long time, it was unclear why the 5′ end of mRNAs would undergo such extensive methylation. Recent studies have revealed that 2′-O methylation of cellular RNA plays a central role in discrimination of self from non-self RNA, e.g., distinction of viral from host RNA [10, 11], while reversible interconversion of N6, 2′-O-dimethyladenosine and 2′-O-methyladenosine in the cap controls mRNA stability .
Not only nucleotides downstream from m7G undergo methylation. In some small nuclear RNAs (snRNAs) involved in splicing of pre-mRNA, the m7G moiety undergoes hypermethylation, i.e., addition of two methyl groups at the N2 position (Fig. 1c). This structure, called a trimethylguanosine cap (TMG cap or m3G), is characteristic of a portion of snRNAs transcribed by RNAP II, namely U1, U2, U4, and U5 snRNAs, and small nucleolar RNAs (snoRNAs) engaged in post-transcriptional modification of precursor ribosomal RNAs (pre-rRNAs) . These snRNAs are hypermethylated by trimethylguanosine synthase 1 (TGS1) after being exported to the cytoplasm in association with Sm proteins (Fig. 4) [13, 14]. The presence of a TMG cap allows binding to the transport protein snurportin 1 (SNP1) and the import of fully matured snRNAs back to the nucleus, where they participate in pre-mRNA splicing . Moreover, the TMG cap has been found at the 5′ end of a certain pool of mRNAs in nematodes (e.g., Caenorhabditis elegans) , and most recently at the 5′ end of mRNAs encoding selenoproteins in mammals .
Interestingly, other snRNAs, such U6 and 7SK, which are synthesized by RNA polymerase III (RNAP III), possess at their 5′ end another modification, a methyl group added directly to triphosphate bridge to generate a γ-methyl phosphate cap (Figs. 1c, 4) . However, the presence of a methyl group linked directly to a triphosphate bridge is not a general feature of RNAP III-transcribed RNAs (e.g., tRNAs), as usually the triphosphorylated 5′ ends of nascent transcripts are trimmed by nucleases to RNAs without cap-like structures upon maturation.
Although it has been hypothesized for a long time that the presence of the 5′ cap is one if the key structural features distinguishing eukaryotic from prokaryotic RNAs, recent mass spectrometry-aided studies on bacterial transcriptomes revealed that in prokaryotes, the 5′ ends of some portion of RNA are modified by moieties, which could be considered as cap-like structures. These include a nicotinamide adenine dinucleotide (NAD+) , 3′-dephospho-coenzyme A (dpCoA) (Fig. 1d) , and other moieties attached to the 5′ end of RNA by an oligophosphate bridge. The structure, biosynthesis, function, and degradation of these so-called bacterial caps have currently come under intense investigation [19, 20, 21, 22].
1.3 Recognition of Cap Structures by Proteins
2 Chemically and Enzymatically Labeled RNA Caps
2.1 Utility of Labeled Capped RNAs and Cap Analogs
Nowadays, capped RNAs can be easily obtained on a small scale by standard molecular biology techniques and used for the purpose of RNA-driven gene expression of proteins of interest in living cells or cell lysates . Variously modified cap analogs are used as small molecule binding and activity probes for cap-binding proteins and cap-processing enzymes. Finally, capped RNAs and small molecule cap analogs are utilized to study RNA turnover in various biological systems. For biochemical studies, capped RNAs, cap analogs, and their degradation products isolated from biological mixtures are often resolved by thin layer chromatography (small nucleotides) or electrophoresis (oligos and nucleic acids) and visualized by different methods. Because of low concentrations of RNA and cap metabolites in typical samples, standard visualization techniques, such as UV shadowing, do not provide sufficient sensitivity nor selectivity in detection, which impairs analysis of complex nucleotide and nucleic acid mixtures. Although fluorescent staining reagents are used to increase sensitivity of nucleic acid detection, they are not useful for detection of short oligos and small nucleotides. Therefore, methods for generation of specifically labeled RNAs carrying radioactive, fluorescent, and other spectroscopic labels are constantly being developed to provide highly sensitive and selective tools for detection of the molecule of interest in a complex mixture or to selectively monitor a particular enzymatic reaction or binding event. In the following sections, we will review enzymatic, chemoenzymatic, and chemical approaches enabling RNA cap labeling, with the main emphasis on modifications of the triphosphate chain and their utility in studying RNA-related processes. We start from the well-established enzymatic approaches, which rely on the use of radioactively labeled Nucleoside triphosphates (NTPs), and RNA-processing enzymes. We then discuss chemical approaches to the synthesis of phosphate-modified or -labeled cap analogs, followed by examples of their utility as molecular probes for studying cap-related processes and reagents for modification of RNA oligonucleotides and long transcripts.
2.2 Cap Biosynthesis
The biosynthesis of other eukaryotic cap structures usually relies on post-transcriptional RNA modification. TMG-capped RNA (m3GpppRNA) is produced by methylation of m7GpppRNA with TGS1 methyltransferase, which acts in vivo on immature U1, U2, U4, and U5 snRNAs, whereas the γ-methyl cap (CH3pppRNA) is generated by methylation of U6 5′-triphosphate snRNA with γ-MTase (Fig. 4). Similarly, it was first proposed that biosynthesis of NAD-capped RNAs in bacteria occurs through the post-synthetic addition of a nicotinamide nucleotide to the 5′ end of RNA [21, 32], but recent studies indicate that this kind of ‘capping’ is rather carried out by bacterial RNA polymerases, which use NAD as a transcription initiator .
2.3 Possible Sites for Cap Modification
From the bioorganic chemist’s point of view, the structural studies provide insights not only into the recognition mode of natural cap structures but also hints at the design of chemically modified cap analog-based molecular tools tailored for specific applications. In general, modifications of the cap should serve at least one of three purposes: (i) to perturb or even completely destroy biological activity (e.g., inhibition of degradation by decapping enzymes); (ii) to increase affinity to particular cap-binding proteins, thereby augmenting associated biological activity; (iii) to confer a new property to the cap structure without any interference with its biological activity (biorthogonal modifications).
Sites for biorthogonal modifications of the cap could be envisaged based on their crystal structure in complex with a targeted cap-binding protein (Fig. 3). For example, in the case of eIF4E complex, both m7G hydroxyl groups and the second nucleoside are exposed to the solvent, and, in fact, these groups were successfully functionalized with various substituents without significant decrease in affinity constant. Consequently, the translational activity of mRNAs carrying such cap structures was at least retained [36, 37, 38, 39]. In contrast, ribose of m7G in complex with DcpS is engaged in numerous contacts and any modification within 2′-O and 3′-O positions results in a considerable decrease of the rate of hydrolysis catalyzed by DcpS [40, 41]. Importantly, the triphosphate bridge appears to be an attractive site for modification, especially in the context of conferring resistance to decapping enzymes. Because the decapping enzymes involved in 5′–3′ and 3′–5′ decay cleave the triphosphate chain at different positions (α,β- and β,γ-, respectively) and employ different mechanisms to perform catalysis, it is possible to develop modifications that affect decapping at only one selected position or at both positions.
2.4 32P Labeling of RNA Caps by Enzymatic Approaches
Capped RNAs and cap analogs radiolabeled with 32P were one of the first tools that enabled biochemical studies of the structural details, functions, and metabolism of mRNA and snRNA caps. One of the most important applications of such tools is to investigate the biochemical activities of cap-specific enzymes involved in RNA turnover. Synthesis of 32P-labeled capped mRNA is usually achieved by transcription of a DNA template in vitro, followed by post-transcriptional enzymatic capping using radiolabeled NTP in at least one of the synthetic steps. Knowing the mechanistic details of phosphate transfer reactions taking place during transcription and capping reactions allows the design of methods to produce transcripts that are site-specifically radiolabeled within or in the vicinity of the cap attached to either a uniformly radiolabeled or unlabeled RNA body.
2.4.1 Incorporation of 32P into RNA Caps
32P labeling can be performed in vitro to create RNA of a pre-determined sequence, but can also be applied to the synthesis of total RNA in cell extracts followed by biochemical analysis. The latter approach has been employed a number of times in different organisms and on various RNA types to analyze the RNA 5′ end heterogeneity, with the focus on both the distal cap modification and the nucleosides at the positions +1 and +2. To this end, total RNA is transcribed in cell extract in the presence of one or more ‘hot’ NTPs. The transcripts are resolved by electrophoresis, fractions of interest excised from the gel, and subjected to degradation by 3′–5′ nuclease such as RNase P1 or a cocktail of ribonucleases, optionally followed by alkaline phosphatase, which releases nucleosides and inorganic phosphate from the RNA body but leaves undigested RNA 5′ ends of Xppp(Np) n (where n = 1–3). Released radioactive products can be analyzed using 2D TLC, usually on cellulose plates, or high-performance liquid chromatography (HPLC) on reversed-phase or ion exchange columns. Alternatively, RNA cleavage can be performed with a more specific endonuclease. For instance, RNase T1 cleaves RNA after 3′-guanylic residues leaving a 3′-phosphate and a 5′-OH in the products. This RNase is particularly useful for the analysis of transcripts initiated with G and labeled within the first transcribed phosphodiester bond (XpppGp*N-RNA), because it cleaves them to release radiolabeled cap structures of XpppGp* type (along with other RNA fragments). The identity of the cap structures can be confirmed by isolation and further digestion by a pyrophosphatase with broad specificity, such as tobacco acid pyrophosphatase (TAP), which cleaves both triphosphate bonds. This approach has been used to identify 5′ ends of eukaryotic and viral mRNAs, snRNAs, and snoRNAs [42, 43, 44, 45], as well as to verify the efficiency of incorporation and orientation of synthetic dinucleotide cap structures introduced into RNA co-transcriptionally [36, 46, 47].
2.4.2 32P-Labeled RNAs and Cap Analogs in the Study of RNA Turnover
Another important use of radiolabeled capped RNAs is for gaining new insights into mRNA and cap turnover. Cap turnover starts when a transcript is subjected to cellular RNA degradation machinery. There are two main mRNA degradation pathways 5′–3′ and 3′–5′, both initiated by poly(A) tail shortening . Although decapping is not the first event in the 5′–3′ mRNA degradation pathway, as it is preceded by deadenylation, it is considered as the first highly irreversible step. In eukaryotes, Dcp1/2 serves as the main decapping enzyme . Dcp2 is a catalytic subunit belonging to the Nudix family of hydrolases [50, 51] whereas Dcp1 is a regulatory subunit, which can interact with additional decapping enhancers such as Edc1, 2, and 3, or PNRC2 .
In contrast, the second main enzyme engaged in cap turnover—DcpS, which is responsible for depletion of products of 3′–5′ RNA decay—prefers cap dinucleotides as substrates . m7Gp*ppN dinucleotides and short m7Gp*pp-RNAs are hydrolyzed by recombinant DcpS between β and γ phosphates to yield m7Gp* and a downstream nucleoside or RNA 5′-diphosphate [57, 58, 59].
Both decapping pathways produce either m7GMP or m7GDP mononucleotides as reaction products. For a long time, it was hypothesized that cells must have developed mechanisms that protect them from accumulation of m7G nucleotides, which either could inhibit cap-dependent proteins or, after conversion into m7GTP, be erroneously salvaged by RNA polymerases. A study on degradation of enzymatically generated m7Gp*ppG-RNA and m7Gp*p in mammalian cell extracts using TLC and autoradiography suggested that an additional function of DcpS in mRNA decay is to cleave m7GDP to m7GMP . However, it was later shown that m7GDP, in contrast to m7GpppG and m7GTP, is not a substrate for DcpS, and at least in vitro acts as a DcpS inhibitor . A new study performed in yeast and mammalian cell extracts on m7Gp*ppG, m7Gp*p, and m7Gp* revealed that m7GDP metabolism may proceed through its phosphorylation to m7GTP carried out by nucleoside diphosphate kinase (NDPK) and subsequent degradation by DcpS to m7GMP . Interestingly, m7GMP is not the end product of cap metabolism in yeast, as degradation of m7Gp*ppG led to only small amounts of m7Gp* and high amounts of radioactive Pi and some unidentified metabolite X with TLC mobility similar to Pi . This study has also shown that in the absence of DcpS activity (gene knockout), the Fhit enzyme can take part in m7Gp*ppG turnover by cleaving it between α and β phosphates to release m7Gp*p . Even less is known about m7GMP metabolism, although a cytosolic nucleotidase III-like enzyme (cNIII) has been described that utilizes m7GMP as a substrate (Fig. 6) .
32P-labeled capped RNA oligonucleotides were used to evaluate the susceptibility of various synthetic cap structures modified in the triphosphate bridge (discussed in more detail below) to Dcp2 [64, 65] and to screen potential inhibitors of Dcp2-catalyzed decapping . If short capped RNA oligonucleotides (up to 50 nt) were used as Dcp2 substrates, the electrophoretic resolution of capped and decapped RNAs could be performed directly on sequencing gels.
2.4.3 Enzymatic Labeling Beyond Radioactive Phosphates
As described in the previous section, by taking advantage of enzymes engaged in cap biosynthesis and turnover, it is possible to generate 32P-labeled versions of variously capped RNAs, different cap structures, and cap metabolites, which provide tools instrumental in numerous biological studies (Fig. 6). However, labeling with 32P has some limitations such as the production of unpredicted radioactive metabolites or incompatibility with continuous (on-line) enzymatic activity monitoring, in vivo studies, and in cellulo visualization. Therefore, labeling methods directed towards introduction of fluorescent labels or biological tags into capped RNA are constantly being developed. Interestingly, some of those methods rely on the same biochemical approaches as those developed for radioactive labeling. For example, it has been recently reported that VCE apart from GTP accepts certain GTP analogs as substrates, including those functionalized at the ribose moiety [67, 68, 69]. Taking advantage of this, VCE has been independently used to transfer a biotinylated  or anthraniloylated  GMP moiety to the 5′ end of RNA from a GTP analog appropriately functionalized at the ribose moiety. Notably, the label structure is of great importance for success in this approach, since a similar reaction using GTP carrying a manthraniloyl dye failed to produce capped transcripts . Another interesting chemo-enzymatic approach to enzymatic cap labeling proposed by Rentmeister et al. takes advantage of methyltransferase activity of trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2-Var1). GlaTgs2-Var1 was first mutated (V34A) to accommodate bulkier, chemically modified analogs of S-adenosyl-l-methionine (SAM) as co-substrates [70, 71, 72], thereby allowing for transfer of various functional groups from synthetic SAM analogs onto the N 2 position of an m7G moiety within the cap. The introduced functional groups were reactive in so-called click reactions such as copper(I) (CuAAC) or strain promoted azide-alkyne cycloaddition (SPAAC), tetrazole photoclick (PC), thiol-ene conjugation (TEC) and inverse electron-demand Diels–Alder cycloaddition (IEDDA) and, as such, were used to label a dinucleotide 5′ cap analog m7GpppA with properly functionalized fluorescent dyes. Although such modifications of N 2 position are expected to disturb mRNA activity in translation, they have great potential for applications related to labeling and quantification of endogenous or exogenously delivered RNAs in vitro and in vivo. Recently, another methyltransferase—Ecm1 from the microsporidian parasite Encephalitozoon cuniculi—was shown to transfer some bulky residues from SAM analogs to the N 7 atom of GpppG capped transcripts . Such functionalized mRNAs were then delivered into HeLa cells by transfection where they reacted with dibenzocyclooctyne functionalized sulforhodamine B in a SPAAC reaction.
2.5 Chemical and Chemoenzymatic Approaches Towards Phosphate-Modified Cap Analogs
2.5.1 Possible Sites for Chemical Modifications of the RNA Caps
The repertoire of available enzymatic methods to modify cap structures is restricted by substrate specificity of particular enzymes. Much greater versatility of cap analogs can be generated by means of chemical synthesis. The influence of chemical modifications within the cap on its biological properties has been studied almost since the very moment of cap discovery. To date, almost every position of m7G and TMG cap structures has been chemically modified, and the influence of those modifications on interaction with cap-binding proteins has been studied, often followed by the determination of the biological consequences of such modifications on RNA stability, transport, and translation. Interestingly, triphosphate chain modifications were explored much later than modifications of the 7-methylguanine or ribose moieties [36, 73, 74, 75, 76], but turned out to have great potential to modulate the interactions of the cap-structure with cap-binding proteins and its susceptibility to different decapping enzymes.
In the next paragraphs, we describe the variety of phosphate modifications that can be introduced into mRNA cap analogs by means of chemical synthesis followed by their selected biochemical, biological and even medicinal applications, as small molecules, as a part of RNA oligonucleotides, and as full-length transcripts.
2.5.2 Synthesis of RNA Caps: P-Imidazolides and MCl2-Mediated Coupling
An important limitation of this method lies in the synthesis of P-imidazolides, which is very efficient for non-modified mono-, di-, and even triphosphates but fails to produce a satisfactory yield of the desired product from compounds modified within the terminal phosphate. A solution to this problem was proposed by introducing electrophilic phosphorylating reagents, such as cyanoethyl phosphate or thiophosphate P-imidazolides, which are capable of reacting with non-activated nucleotides (Fig. 7) . Such reagents were shown to react with a series of phosphates, phosphorothioates, and seleno- and borano-phosphates, which was followed by one-step removal of cyanoethyl-protecting groups, provided straightforward access to α-modified nucleoside diphosphates and β-modified triphosphates.
Another example of the usefulness of MCl2-mediated coupling reactions involving P-imidazolides is the synthesis of a symmetrical two-headed tetraphosphate cap analog , which was found to be a potent inhibitor of Dcp2 decapping enzyme acting as an mRNA 5′ cap mimic [27, 66]. Owing to the symmetry of the final compound, it was possible to perform two coupling reactions in one synthetic step, starting with P1,P2-diimidazolyl-pyrophosphate and two equivalents of 7-methylguanosine 5′-phosphorothioate (Fig. 8b).
MCl2-mediated coupling reactions usually reach very high conversion of reactants in a reasonable length of time (1–48 h depending on the sterical hindrance); however, they require an excess of divalent metal chloride (4–16 molar equivalents) [77, 78]. ZnCl2, MgCl2, or MnCl2 are the most commonly used for that purpose (Fig. 8). The role of metal ions is probably complex and has never been studied in detail, but the catalytic effect could be mainly attributed to three aspects related to the formation of phosphate complexes (Fig. 8d): (i) eliminating the electrostatic repulsion between negatively charged moieties and templating the reactants, (ii) increasing the solubility of reactants in an organic solvent (usually DMF), and (iii) increasing the electrophilicity of phosphorus atoms by complexation of imidazole.
2.5.3 Phosphate-Modified Cap Analogs as Molecular Probes for Cap-Dependent Processes
2.5.4 mRNA Modification with Synthetic Cap Analogs (Transcription In Vitro)
Beside their use as small molecular ligands, phosphate-modified dinucleotide cap analogs can be used as reagents for mRNA 5′ end modification, as previously reviewed by us in detail .
Importantly, the co-transcriptional capping approach opens the possibility for straightforward introduction of chemical modifications into the cap. First, the ‘anti-reverse’ type modifications of the m7G cap can be additionally functionalized to carry a label or a biological tag such as biotin or fluorescent dye. Second, a number of phosphate-modified nucleotides were shown to be accepted as transcription initiators for T7 and SP6 RNA polymerases, giving access to phosphate-modified capped mRNAs as tools to study the specificity of cleavage by RNA-dependent capping enzymes, with Dcp1/2 being the prime example [64, 65, 108]. Two methylene-bisphosphonate ARCAs differing in the site of O to CH2 substitution (either α–β or β–γ, Fig. 9) allowed for differential inhibition of decapping pathways . First, in vitro experiments were performed on short radiolabeled RNAs to determine the susceptibility of the modified caps to recombinant hDcp2. It was found that the RNAs with the cap modified at the α–β position were resistant to cleavage by Dcp2, while the RNAs with the cap modified at the β–γ position were similarly susceptible to the unmodified cap. A study on degradation of full-length mRNAs in mammalian cells revealed that preventing the cleavage at the α–β position of the triphosphate bridge (performed by Dcp1/2) increases the half-life of mRNA. This observation combined with earlier studies which showed that modification at the β-γ position protects the cap from cleavage only by DcpS leads to the conclusion that cap degradation is a limiting step for the 5′–3′ degradation pathway but not for the 3′–5′ pathway (Fig. 9).
Despite the extended half-life of mRNAs capped with α-β modified analogs, such transcripts had poor translational properties, which inspired the quest for novel cap structures that could confer to mRNA both resistance to Dcp2 and high translational efficiency. The first analogs, which combined these two features, were compounds carrying a non-bridging O–S substitution at the beta position of the triphosphate chain, referred to as β-S-ARCAs . Later, it was found that similar properties could be conferred to mRNA by an O-to-BH3 substitution [65, 83]. Recent studies have shown that applying two O–S substitutions at two adjacent phosphate moieties in a tri- or tetraphosphate bridge may provide biological properties superior to β-S-ARCA . A series of phosphate-modified analogs carrying O-to-S, O-to-NH, O-to-BH3, and O-to-Se substitutions at different positions have been used to study mRNA degradation in HeLa cells .
Typically, even small bridging modifications of the oligophosphate chain decrease the translation potential of mRNA, but the relation between size of the substituent and biological properties is not so obvious. Recently, a series of 34 cap analogs carrying a 1,2,3-triazole moiety within the oligophosphate chain were synthesized using copper-catalyzed azide-alkyne cycloaddition . Biochemical evaluation of RNA co-transcriptionally capped with these analogs led to identification of two analogs, which despite this bulky triazole modification had translational properties similar to cap structures carrying an unmodified triphosphate chain. This unexpected finding highlights possibilities of novel approaches towards the synthesis of small molecule cap analogs as well as capped RNAs.
2.5.5 Phosphate Modifications for Chemical Capping Approaches
Over the 40 years since the discovery of the cap structure, chemical synthesis has provided access to a still-growing library of small molecule cap analogs, while the molecular biology techniques enabled incorporation of these structures into long RNAs. Somewhat surprisingly, exploring the ‘middle ground’—which is the efficient synthesis of short capped oligonucleotides in high purity—still poses a challenge both for chemistry and biology. Short m7GpppRNAs, m3GpppRNAs, and NAD-RNAs have been synthesized by chemical reaction of RNA-5′ phosphates with appropriate imidazole-activated nucleotides under aqueous conditions [109, 110, 111, 112], but these reactions are only moderately efficient and require time-consuming chromatographic and/or enzymatic work-ups to separate capped and uncapped RNAs. An improvement in solution synthesis of capped oligomers has been proposed by using a 4,4′-dimethoxytrityl (DMT) group as a lipophilic purification handle, which facilitates separation of capped RNAs from uncapped RNAs by RP HPLC . Solid-phase synthesis of capped oligonucleotides has been attempted as well, but found to be challenging due to incompatibility of the m7G moiety with standard oligonucleotide de-immobilization and de-protection protocols [114, 115, 116, 117, 118]. Combination of RNA capping via these approaches with phosphate modifications appears even more challenging and remains to be demonstrated.
3 Summary and Future Prospects
Cap is a natural tag attached to the 5′ end of messenger RNA and small nuclear RNA, which due to its unique structural features fulfills essential biological functions during the process of gene expression. The ‘inverted’ 5′-to-5′ triphosphate bridge, alongside with a 7-methylguanosine moiety, is one of the two crucial elements of the mRNA cap structure. The triphosphate bridge is a site of cleavage for RNA decapping enzymes and is involved in the electrostatic interactions with other cap-related proteins. Therefore, synthetic cap analogs modified within the triphosphate bridge or carrying spectroscopic labels attached to phosphate moieties turned out to be valuable tools for studies on mRNA metabolism, modification of natural mRNA properties, or molecular probes for screening inhibitors of cap-dependent processes/proteins. Biorthogonal methods for modification of biologically relevant molecules give exceptional opportunities to follow or even to manipulate these processes inside the cells. mRNA has a great potential for use as a therapeutic agent in gene therapy, emerging in several ongoing clinical trials, which is a driving motivation for the development of new molecular tools for mRNA cap modification and for the construction of new cap-based molecular probes. Much progress has been made in microscopic techniques and single-molecule experiments allowing investigation into the properties of individual molecules, and providing another motivation to develop new methods for mRNA cap-labeling. The authors of this chapter are convinced that the coming years will bring intensification of studies aimed at developing new applications of labeled cap analogs in a cellular context. The continuous improvements in understanding of how the cap interacts with its cellular partners, protein factors, and decapping enzymes lead us to believe that in the future, modifications introduced into the mRNA cap structure will minimize the interruption of mRNA biological functions. Progress in the design and synthesis of molecular probes will enable investigation of processes that are poorly understood, such as cellular correlation between initiation of translation and mRNA degradation, post-transcriptional methylation of mRNA 5′ end, and antiviral immune response. The discovery of structures similar to the mRNA cap, such as the NAD cap in bacteria, may also open a new chapter in this field. Various approaches applied for the investigation of mRNA and snRNA caps can be adapted to study biological functions of their bacterial relatives. Finally, advancements in screening methodologies will allow researchers to find more selective inhibitors of cap-dependent processes with high therapeutic potency. The immense progress in nucleotide and nucleic acid delivery methodologies we are currently witnessing will likely bring phosphate-modified cap analogs to in vivo applications.
This work was financially supported by the National Science Centre (Poland, grants UMO-2013/09/B/ST5/01341 to JJ and UMO-2011/01/D/ST5/05869 to JK) and by the Ministry of Science and Higher Education (Poland, DI2012 024842 to MW).
- 5.Furuichi Y, Shatkin AJ (2000) Advances in virus research, vol 55. Academic Press Inc, San Diego, pp 135–184Google Scholar
- 17.Gupta S, Busch RK, Singh R, Reddy R (1990) Characterization of U6 small nuclear-RNA cap-specific antibodies: identification of gamma-monomethyl-GTP cap structure in 7SK and several other human small RNAs. J Biol Chem 265:19137–19142Google Scholar
- 24.Niedzwiecka A, Marcotrigiano J, Stepinski J, Jankowska-Anyszka M, Wyslouch-Cieszynska A, Dadlez M, Gingras AC, Mak P, Darzynkiewicz E, Sonenberg N et al (2002) Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5′ cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. J Mol Biol 319:615–635CrossRefGoogle Scholar
- 34.Langberg SR, Moss B (1981) Post-transcriptional modifications of messenger-RNA - purification and characterization of cap-I and cap-II RNA nucleoside-2′-O-methyltransferases from HeLa-cells. J Biol Chem 256:54–60Google Scholar
- 35.Shuman S, Surks M, Furneaux H, Hurwitz J (1980) Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions: association of the GTP-pyrophosphate exchange activity with vaccinia messenger-RNA guanylyltransferase.RNA (guanine-7-)methyltransferase complex (capping enzyme). J Biol Chem 255:1588–1598Google Scholar
- 36.Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001) Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG. RNA-a Publ RNA Soc 7:1486–1495Google Scholar
- 37.Warminski M, Kowalska J, Buck J, Zuberek J, Lukaszewicz M, Nicola C, Kuhn AN, Sahin U, Darzynkiewicz E, Jemielity J (2013) The synthesis of isopropylidene mRNA cap analogs modified with phosphorothioate moiety and their evaluation as promoters of mRNA translation. Bioorg Med Chem Lett 23:3753–3758CrossRefGoogle Scholar
- 45.Shimba S, Buckley B, Reddy R, Kiss T, Filipowicz W (1992) Cap structure of U3 small nucleolar RNA in animal and plant-cells is different: gamma-monomethyl phosphate cap structure in plant RNA. J Biol Chem 267:13772–13777Google Scholar
- 46.Pasquinelli AE, Dahlberg JE, Lund E (1995) Reverse 5′ caps in RNAs made in vitro by phage RNA polymerases. RNA-a Publ RNA Soc 1:957–967Google Scholar
- 61.Wypijewska A, Bojarska E, Lukaszewicz M, Stepinski J, Jemielity J, Davis RE, Darzynkiewicz E (2012) 7-Methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity. Biochemistry 51:8003–8013CrossRefGoogle Scholar
- 65.Su W, Slepenkov S, Grudzien-Nogalska E, Kowalska J, Kulis M, Zuberek J, Lukaszewicz M, Darzynkiewicz E, Jemielity J, Rhoads RE (2011) Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH3, Se, and NH. RNA-a Publ RNA Soc 17:978–988CrossRefGoogle Scholar
- 68.Gunawardana D, Domashevskiy AV, Gayler KR, Goss DJ (2016) Efficient preparation and properties of mRNAs containing a fluorescent cap analog: anthraniloyl-m7G pppG (vol 3, e988538, 2014). Translation 4:1Google Scholar
- 69.Ettwiller L, Buswell J, Yigit E, Schildkraut I (2016) A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genom 17, 14, No 199Google Scholar
- 73.Darzynkiewicz E, Stepinski J, Ekiel I, Goyer C, Sonenberg N, Temeriusz A, Jin YX, Sijuwade T, Haber D, Tahara SM (1989) Inhibition of eukaryotic translation by nucleoside 5′-monophosphate analogs of messenger RNA 5′-cap: changes in N7 substituent affect analog activity. Biochemistry 28:4771–4778CrossRefGoogle Scholar
- 78.Kadokura M, Wada T, Urashima C, Sekine M (1997) Efficient synthesis of gamma-methyl-capped guanosine 5′-triphosphate as a 5′-terminal unique structure of U6 RNA via a new triphosphate bond formation involving activation of methyl phosphorimidazolidate using ZnCl2 as a catalyst in DMF under anhydrous conditions. Tetrahedron Lett 38:8359–8362CrossRefGoogle Scholar
- 80.Kowalska J, Lewdorowicz M, Zuberek J, Grudzien-Nogalska E, Bojarska E, Stepinski J, Rhoads RE, Darzynkiewicz E, Davis RE, Jemielity J (2008) Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS. RNA-a Publ RNA Soc 14:1119–1131CrossRefGoogle Scholar
- 81.Strenkowska M, Grzela R, Majewski M, Wnek K, Kowalska J, Lukaszewicz J, Zuberek J, Darzynkiewicz E, Kuhn AN, Sahin U et al (2016) Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential. Nucl Acid Res 44:9578–9590Google Scholar
- 83.Kowalska J, Wypijewska del Nogal A, Darzynkiewicz ZM, Buck J, Nicola C, Kuhn AN, Lukaszewicz M, Zuberek J, Strenkowska M, Ziemniak M et al (2014) Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes. Nucl Acid Res 42:10245–10264CrossRefGoogle Scholar
- 85.Rydzik AM, Kulis M, Lukaszewicz M, Kowalska J, Zuberek J, Darzynkiewicz ZM, Darzynkiewicz E, Jemielity J (2012) Synthesis and properties of mRNA cap analogs containing imidodiphosphate moiety-fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis. Bioorg Med Chem 20:1699–1710CrossRefGoogle Scholar
- 90.Kuhn AN, Diken M, Kreiter S, Selmi A, Kowalska J, Jemielity J, Darzynkiewicz E, Huber C, Tureci O, Sahin U (2010) Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther 17:961–971CrossRefGoogle Scholar
- 94.Zytek M, Kowalska J, Lukaszewicz M, Wojtczak BA, Zuberek J, Ferenc-Mrozek A, Darzynkiewicz E, Niedzwiecka A, Jemielity J (2014) Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5′,5′-triphosphate bridge. Org Biomol Chem 12:9184–9199CrossRefGoogle Scholar
- 95.Honcharenko M, Bestas B, Jezowska M, Wojtczak BA, Moreno PMD, Romanowska J, Bachle SM, Darzynkiewicz E, Jemielity J, Smith CIE et al (2016) Synthetic m(3)G-CAP attachment necessitates a minimum trinucleotide constituent to be recognised as a nuclear import signal. RSC Adv 6:51367–51373CrossRefGoogle Scholar
- 96.Wojtczak BA, Warminski M, Kowalska J, Lukaszewicz M, Honcharenko M, Smith CIE, Stromberg R, Darzynkiewicz E, Jemielity J (2016) Clickable trimethylguanosine cap analogs modified within the triphosphate bridge: synthesis, conjugation to RNA and susceptibility to degradation. RSC Adv 6:8326–8337CrossRefGoogle Scholar
- 100.Grudzien-Nogalska E, Stepinski J, Jemielity J, Zuberek J, Stolarski R, Rhoads RE, Darzynkiewicz E (2007) Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. Transl Initiat: Cell Biol High-Through Method Cheml Based Approach 431:203–227Google Scholar
- 110.Zuberek J, Wyslouch-Cieszynska A, Niedzwiecka A, Dadlez M, Stepinski J, Augustyniak W, Gingras AC, Zhang ZB, Burley SK, Sonenberg N et al (2003) Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein. RNA-a Publ RNASoc 9:52–61CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.