Preparation of materials for flexizyme reactions and genetic code reprogramming

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus speci c sequences of non-standard peptides can be ribosomally expressed according to their mRNA templates.We have developed a protocol that facilitates the genetic code reprogramming using exizymes integrated with a custom-made in-vitro translation apparatus, referred to as the exible in-vitro translation \(FIT) system.Flexizymes are exible tRNA acylation ribozymes that enable the preparation of a diverse array of non-proteinogenic acyl-tRNAs.These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired non-standard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds.We here describe a protocol for the preparation of DNA templates for exizymes, tRNAs, microhelix RNA, and mRNAs that are used for genetic code reprogramming with exizymes. ReagentsTris\(hydroxymethyl)aminomethane \(Tris), nuclease and protease tested \(Nacalai tesque, cat.no.35434-34) Hydrochloric acid \(35%) \(Nacalai tesque, cat.no.18321-05) \! CAUTION Corrosive; irritant.Wear gloves and protecting glasses.Potassium chloride \(Nacalai tesque, cat.no.28514-75) Triton X-100 \(Nacalai tesque, cat.no.35501-15) Magnesium chloride hexahydrate \(Nacalai tesque, cat.no.20909-55) 100 mM dATP solution \(Nippon gene, custom made) 100 mM dCTP solution \(Nippon gene, custom made) 100 mM dGTP solution \(Nippon gene, custom made) 100 mM dTTP solution \(Nippon gene, custom made) Oligodeoxyribonucleotides \(Operon) Agarose for 50-800 bp fragment \(Nacalai tesque, cat.no.01147-96) Ethidium bromide solution \(10 mg/ml) \(Nacalai tesque, cat.no.14631-94) \! CAUTION Mutagen; irritant.Wear gloves.Acetic Acid \(Kanto chemical, cat.no.01021-00) \! CAUTION Corrosive; irritant.0.5 mol/l-EDTA solution \(pH 8.0) \(Nacalai tesque, cat.no.14347-21) Phenol, saturated with TE buffer \(Nacalai tesque, cat.no.26829-96) \! CAUTION Toxic; corrosive, irritant.Chloroform \(Nacalai tesque, cat.no.08402-55) \! CAUTION Irritant.3-Methyl-1-butanol \(isoamyl alcohol) \(Nacalai tesque, cat.no.02715-45) \! CAUTION Flammable.Ethanol \(Nacalai tesque, cat.no.14713-95) \! CAUTION Flammable.Sodium chloride \(Nacalai tesque, cat.no.31320-05) Dithiothreitol, nuclease tested \(Nacalai tesque, cat.no.14128-04) Spermidine \(Nacalai tesque, cat.no.32108-04) \! CAUTION Corrosive.Adenosine 5'-triphosphate disodium salt n-hydrate \(Wako Pure Chemical Industries, cat.no.133-00725) CTP sodium salt \(JENA Bioscience, cat.no.NU-1011) GTP sodium salt \(JENA Bioscience, cat.no.NU-1012) UTP sodium salt \(JENA Bioscience, cat.no.NU-1013) Potassium hydroxide \(Nacalai tesque, cat.no.28616-45) \! CAUTION Corrosive; irritant.Wear gloves and protecting glasses.Manganese \(II) chloride tetrahydrate \(Wako Pure Chemical Industries, cat.no.133-00725) RQ1 RNase-free DNase \(1 U/μl) \(