Modified Nucleotides for Chemical and Enzymatic Synthesis of Therapeutic RNA

Abstract

In recent years RNA has emerged as a medium with a broad spectrum of therapeutic potential, however, for years, a group of short RNA fragments was studied and considered therapeutic molecules. In nature, RNA plays both functions, with coding and non-coding potential. For RNA, like for any other therapeutic, to be used clinically, certain barriers must be crossed. Among them, there are biocompatibility, relatively low toxicity, bioavailability, increased stability, target efficiency and low off-target effects. In the case of RNA, most of these obstacles can be overcome by incorporating modified nucleotides into its structure. This may be achieved by both, in vitro and in vivo biosynthetic methods, as well as chemical synthesis. Some advantages and disadvantages of each approach are summarized here. The wide range of nucleotide analogues has been tested for their utility as monomers for RNA synthesis. Many of them have been successfully implemented and a lot of pre-clinical and clinical studies involved modified RNA is carried out. Some of these medications have already been introduced into clinics. After the huge success of RNA-based vaccines that were introduced into widespread use in 2020, and the introduction to the market of some RNA-based drugs, RNA therapeutics containing modified nucleotides appear to be the future of medicine.

Bioengineered chimeric tRNA/pre‐miRNAs as prodrugs in cancer therapy

“…miRNAs are mainly synthesized as miRNA mimics in vitro by chemical methods with a wide range of synthetic modifications. In this method, the recombinant T7 RNA polymerase protein from bacteriophage T7 was typically used to generate RNA sequences from DNA templates 64,65 . These molecules had extensive artificial modifications on ribose and phosphate groups (Figure 4a) that could alter their tertiary structure, physicochemical properties, safety profile, and biological and pharmacological activities.…”

“…Synthetic miRNA mimics are produced by automated systems (a), and bioengineered miRNAs can be efficiently produced by various carriers through economic microbial fermentation (b). bacteriophage T7 was typically used to generate RNA sequences from DNA templates 64,65. These molecules had extensive artificial modifications on ribose and phosphate groups (Figure4a) that could alter their tertiary structure, physicochemical properties, safety profile, and biological and pharmacological activities.…”

Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP

“…9 Introduction of modifications into RNA has been commonly used for years to provide higher stability and more effective transport and to enhance the activity of such modified molecules. 5,10 A common way to introduce modifications into the RNA strand is in vitro RNA transcription. Several bacteriophage enzymes such as T7, T3, and Sp6 RNA polymerases have been shown to catalyze the in vitro synthesis of modified RNA molecules.…”

“…3 One of the most important applications of modified nucleotides is also their use as building blocks for the synthesis of DNA and RNA. 4,5 This role of modified nucleotides has recently become particularly important, especially with the widespread utilization of mRNA vaccines to combat the spread of SARS-CoV-2. 6−8 Nowadays, mRNA-based drugs are considered to open a new era of therapeutic approaches.…”

“…These natural counterparts are known as signaling molecules, agonists of receptors, substrates and cofactors of enzymes, as well as the building blocks for RNA synthesis. , Through the multifunctional nature of nucleosides and nucleotides, their analogues are commonly designed and synthesized for the regulation of signaling pathways acting as substrates, inhibitors, cofactors of enzymes, or agonists or antagonists of receptors . One of the most important applications of modified nucleotides is also their use as building blocks for the synthesis of DNA and RNA. , This role of modified nucleotides has recently become particularly important, especially with the widespread utilization of mRNA vaccines to combat the spread of SARS-CoV-2. − Nowadays, mRNA-based drugs are considered to open a new era of therapeutic approaches . Introduction of modifications into RNA has been commonly used for years to provide higher stability and more effective transport and to enhance the activity of such modified molecules. , …”