Editing Cleancap mRNA Invitro Transcription (section)

== UTP&nbsp; ==

Uridine 5'-Triphosphate, Sigma , 94370-1G, 1 g $288

[[File:Uridine-5'-Triphosphate.png|RTENOTITLE]]

Pseudouridine-5'-Triphosphate,&nbsp;Pseudo-UTP, 5-Ribosyl Uracil,&nbsp; N-1019-5,5 µmole $451

Pseudouridine (5-ribosyluracil) was the first modified ribonucleoside discovered. It is the most abundant natural modified RNA base, and has been deemed the "fifth nucleoside" in RNA. It can be found in structural RNAs, such as transfer, ribosomal and small nuclear RNA. Pseudouridine has been found to enhance base stacking and translation. Pseudouridine-5'-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors with in vitro transcribed RNA. Pseudo-UTP, along with 5-methylcytidine-5'-triphosphate (5-methyl-CTP) has shown innate immune suppression in culture and in vivo while enhancing translation in recent publications. For example, Warren et al. determined an efficient means of reprogramming multiple human cell types using modified mRNA that can express the four primary reprogramming proteins. These cells are referred to as induced pluripotency stem cells (iPSCs). Warren et al. found that enzymatically synthesized RNA substituted with Pseudo-UTP, 5-Methyl-CTP and ARCA effectively evaded the cell’s innate immune response, a crucial component in their success. Reduced toxicity due to substitution with Pseudo-UTP, 5-Methyl-CTP was critical since it allowed repeated transfection with in vitro transcribed mRNA over several weeks.

https://www.targetmol.com/compound/mrtx-1719

[[File:Pseudouridine-5'-Triphosphate.png|400px|RTENOTITLE]]

5-Methyluridine-5'-Triphosphate ,&nbsp; 5-Methyl-UTP, 5-Me-UTP, 5-mUTP, Trilink:&nbsp;N-1024-5,5 µmole $464.

Thermostability Enhancer

[[File:5-Methyluridine-5'-Triphosphate.png|RTENOTITLE]]

N1-Methyl-Pseudouridine-5'-Triphosphate,&nbsp;N1-Methylpseudo-UTP, 1-Methylpseudo-UTP, Trilink: N-1081-5,5 µmole $464

Incorporation of N1-methylpseudouridine can reduce the immunogenicity of the resulting mRNA

[[File:N1-Methyl-Pseudouridine-5'-Triphosphate.png|RTENOTITLE]]

https://www.targetmol.com/compound/uridine_5_triphosphate_tetrasodium

'''Reagents not supplied:'''

RiboMAX™ Large Scale RNA Production Systems – T7: Promega P1300<br/>ScriptCap™ m7G Capping System: CellScript C-SCCE0625<br/>ScriptCap™ 2'-O-Methyltransferase Kit: CellScript C-SCMT0625<br/>A-Plus™ Poly(A) Polymerase Tailing Kit: CellScript C-PAP5104H<br/>Antarctic Phosphatase: New England BioLabs M0289S<br/>ScriptGuard™ RNase Inhibitor: CellScript C-SRI6310K

Lipofectamine™ MessengerMAX™ Transfection Reagent: ThermoFisher LMRNA001<br/>RiboJuice™ mRNA Transfection Kit: MilliporeSigma TR-1013