Chemical Modifications of mRNA Ends for Therapeutic Applications.

Messenger ribonucleic acid (mRNA) is the universal cellular instruction for ribosomes to produce proteins. Proteins are responsible for most of the functions of living organisms, and their abnormal structure or activity is the cause of many diseases. mRNA, which is expressed in the cytoplasm and, unlike DNA, does not need to be delivered into the nucleus, appears to be an ideal vehicle for pursuing the idea of gene therapy in which genetic information about proteins is introduced into an organism to exert a therapeutic effect. mRNA molecules of any sequence can be synthesized using the same set of reagents in a cell-free system via a process called in vitro transcription (IVT), which is very convenient for therapeutic applications. However, this does not mean that the path from the idea to the first mRNA-based therapeutic was short and easy. It took 30 years of trial and error in the search for solutions that eventually led to the first mRNA vaccines created in record time during the SARS-CoV-2 pandemic. One of the fundamental problems in the development of RNA-based therapeutics is the legendary instability of mRNA, due to the transient nature of this macromolecule. From the chemical point of view, mRNA is a linear biopolymer composed of four types of ribonucleic subunits ranging in length from a few hundred to hundreds of thousands of nucleotides, with unique structures at its ends: a 5'-cap at the 5'-end and a poly(A) tail at the 3'-end. Both are extremely important for the regulation of translation and mRNA durability. These elements are also convenient sites for sequence-independent labeling of mRNA to create probes for enzymatic assays and tracking of the fate of mRNA in cells and living organisms. Synthetic 5'-cap analogs have played an important role in the studies of mRNA metabolism, and some of them have also been shown to significantly improve the translational properties of mRNA or affect mRNA stability and reactogenicity. The most effective of these is used in clinical trials of mRNA-based anticancer vaccines. Interestingly, thanks to the knowledge gained from the biophysical studies of cap-related processes, even relatively large modifications such as fluorescent tags can be attached to the cap structure without significant effects on the biological properties of the mRNA, if properly designed cap analogs are used. This has been exploited in the development of molecular tools (fluorescently labeled mRNAs) to track these macromolecules in complex biological systems, including organisms. These tools are extremely valuable for better understanding of the cellular mechanisms involved in mRNA metabolism but also for designing therapeutic mRNAs with superior properties. Much less is known about the usefulness/utility of poly(A) tail modifications in the therapeutic context, but it is clear that chemical modifications of poly(A) can also affect biochemical properties of mRNA. This Account is devoted to chemical modifications of both the 5'- and 3'-ends of mRNA aimed at improving the biological properties of mRNA, without interfering with its translational function, and is based on the authors' more than 20 years of experience in this field.

Preparation of RNAs with non-canonical 5' ends using novel di- and trinucleotide reagents for co-transcriptional capping.

Many eukaryotic and some bacterial RNAs are modified at the 5' end by the addition of cap structures. In addition to the classic 7-methylguanosine 5' cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to in vitro transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations. To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5'-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5' capping.

2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between 'self' and 'non-self' in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2'-O-methylation of the second transcribed nucleotide within the mRNA 5' cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2'-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as 'self' and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.

RNA Ligation for Mono and Dually Labeled RNAs.

Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3'-end labeling of in vitro transcribed RNAs. We synthesized novel adenosine 3',5'-bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide-containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain-promoted alkyne-azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD-capped RNAs, and 5'-fluorescently labeled m7 Gp3 Am -capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence-based monitoring of decapping. This method will facilitate the use of various functionalized mRNA-based probes.

Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation.

Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein-protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.

NAD Analogs in Aid of Chemical Biology and Medicinal Chemistry.

Nicotinamide adenine dinucleotide (NAD) serves as an essential redox co-factor and mediator of multiple biological processes. Besides its well-established role in electron transfer reactions, NAD serves as a substrate for other biotransformations, which, at the molecular level, can be classified as protein post-translational modifications (protein deacylation, mono-, and polyADP-ribosylation) and formation of signaling molecules (e.g., cyclic ADP ribose). These biochemical reactions control many crucial biological processes, such as cellular signaling and recognition, DNA repair and epigenetic modifications, stress response, immune response, aging and senescence, and many others. However, the links between the biological effects and underlying molecular processes are often poorly understood. Moreover, NAD has recently been found to tag the 5'-ends of some cellular RNAs, but the function of these NAD-capped RNAs remains largely unrevealed. Synthetic NAD analogs are invaluable molecular tools to detect, monitor, structurally investigate, and modulate activity of NAD-related enzymes and biological processes in order to aid their deeper understanding. Here, we review the recent advances in the design and development of NAD analogs as probes for various cellular NAD-related enzymes, enzymatic inhibitors with anticancer or antimicrobial therapeutic potential, and other NAD-related chemical biology tools. We focus on research papers published within the last 10 years.

Straightforward Ball-Milling Access to Dinucleoside 5',5'-Polyphosphates via Phosphorimidazolide Intermediates.

A solvent-assisted mechanochemical approach to access symmetrical and mixed dinucleoside 5,5'-polyphosphates is reported. Under ball-milling conditions, nucleoside 5'-monophosphates were quantitatively activated using 1,1'-carbonyldiimidazole, forming their phosphorimidazolide derivatives. The addition of a nucleoside 5'-mono-, di- or triphosphate directly led to the formation of the corresponding dinucleotides. Benefits of the reported one-pot method include the use of unprotected nucleotides in their sodium or acid form, activation by the eco-friendly 1,1'-carbonyldiimidazole, non-dry conditions, short reaction time, high conversion rates, and easy setup and purification. This work offers new perspectives for the synthesis of nucleotide conjugates and analogues, combining the phosphorimidazolide approach and milling conditions.

Nicotinamide-Containing Di- and Trinucleotides as Chemical Tools for Studies of NAD-Capped RNAs.

We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.

Water-Medium Synthesis of Nucleoside 5'-Polyphosphates.

This unit describes a one-pot, two step synthesis of ribonucleoside 5'-di- and 5'-triphosphates, as well as their purification. The first step of the synthesis involves the activation of an unprotected ribonucleoside 5'-monophosphate with 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate and imidazole, in a mixture of water/acetonitrile. The resulting phosphorimidazolate intermediate is then treated with inorganic phosphate or pyrophosphate to afford the corresponding nucleoside 5'-di- or 5'-triphosphates. The attractive features of this strategy include the absence of protecting groups on the starting material and convenient set up (i.e., use of water, non-dry solvents and reagents, commercially available sodium salts). © 2017 by John Wiley & Sons, Inc.

Recent Trends in Nucleotide Synthesis.

Focusing on the recent literature (since 2000), this review outlines the main synthetic approaches for the preparation of 5'-mono-, 5'-di-, and 5'-triphosphorylated nucleosides, also known as nucleotides, as well as several derivatives, namely, cyclic nucleotides and dinucleotides, dinucleoside 5',5'-polyphosphates, sugar nucleotides, and nucleolipids. Endogenous nucleotides and their analogues can be obtained enzymatically, which is often restricted to natural substrates, or chemically. In chemical synthesis, protected or unprotected nucleosides can be used as the starting material, depending on the nature of the reagents selected from P(III) or P(V) species. Both solution-phase and solid-support syntheses have been developed and are reported here. Although a considerable amount of research has been conducted in this field, further work is required because chemists are still faced with the challenge of developing a universal methodology that is compatible with a large variety of nucleoside analogues.