Variety of Nucleotide Polymerase Mutants Aiming to Synthesize Modified RNA

Abstract

Significant efforts have been made to develop therapeutic RNA aptamers that exploit synthetic RNA to capture target molecules. However, ensuring RNA aptamers are resistant against intrinsic nucleases remains an issue and restricts their use as therapeutics. Introduction of chemical modifications to the 2' sugar moiety of RNA improves their stability effectively and can be achieved by chemical synthesis using modified phosphoramidites; however, this approach is not suitable for preparing long RNA molecules. Although recombinant nucleotide polymerases can transcribe RNA, these polymerases cannot synthesize modified RNA because they do not recognize 2' modified nucleoside triphosphates. In this review, we focus on several polymerase mutants that tolerate substrates containing modifications of the 2' sugar moiety to synthesize RNA, and the problems that must be overcome to prepare chemically modified RNA with high efficacy by in vitro transcription.

Evolution of complexity in non-viral oligonucleotide delivery systems: from gymnotic delivery through bioconjugates to biomimetic nanoparticles

“…Addition of ROS scavengers into LNPs or use of modified lipids at the inner leaflet can alleviate the problem. Alternatively, developing strategies with polymerases that can site-specifically incorporate 2’-modified nucleotides also yield improved stability[ 163 ]. RNA delivery systems dramatically evolved in the last two decades from gymnotic delivery to multimodal, environment-responsive complex formulations.…”

Thermophilic Nucleic Acid Polymerases and Their Application in Xenobiology

“…Thermophilic nucleic acid polymerases and their mutants have been extensively explored and used in the synthesis, reverse transcription, and even amplification of XNAs [ 5 , 135 ]. Although some other polymerases that are not high temperature tolerant, such as mutants of T7 RNAP, have also been used for the synthesis of modified nucleic acids [ 136 ], thermophilic nucleic acid polymerases are indispensable for XNA synthesis, reverse transcription, or amplification at high temperatures or with thermocycling programs, which are essential when tough templates with complex secondary structures are used or are important for higher yields.…”

Aptamer Applications in Emerging Viral Diseases

“…Additionally, post-SELEX modifications to produce 2′-O-methyl aptamers are needed, which might change aptamer structures. However, another group developed nucleotide polymerase mutants, which tolerate modifications at the 2′-OH ribose group and can be readily used for the selection of nuclease-resistant RNA aptamers [ 139 ]. Limitations also account for thioaptamers with phosphorothioate or phosphorodithioate linkages and Spiegelmers, which rely on chemical synthesis procedures [ 140 , 141 ].…”