title: Optimization for in vitro transcription conditions off mRNAs
title: Ð²ÑÐ¿ÑÑÐºÐ½Ð°Ñ ÐºÐ²Ð°Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¾Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¼Ð°Ð³Ð¸ÑÑÑÐ°
titleType: Subtitle
title: ÐÐ¿ÑÐ¸Ð¼Ð¸Ð·Ð°ÑÐ¸Ñ ÑÑÐ»Ð¾Ð²Ð¸Ð¹ ÑÐµÐ°ÐºÑÐ¸Ð¸ in vitro ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ð¸ Ð¼Ð ÐÐ
Creators: name: Ð®ÑÑÐµÐ½ÐºÐ¾, ÐÐ»ÐµÐºÑÐ°Ð½Ð´ÑÐ°
Publisher: name: Ð¡Ð°Ð½ÐºÑ-ÐÐµÑÐµÑÐ±ÑÑÐ³ÑÐºÐ¸Ð¹ Ð¿Ð¾Ð»Ð¸ÑÐµÑÐ½Ð¸ÑÐµÑÐºÐ¸Ð¹ ÑÐ½Ð¸Ð²ÐµÑÑÐ¸ÑÐµÑ ÐÐµÑÑÐ° ÐÐµÐ»Ð¸ÐºÐ¾Ð³Ð¾
Year: 2023
Subjects: ÐÐ¸Ð¾ÑÐµÑ Ð½Ð¾Ð»Ð¾Ð³Ð¸Ñ
ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ñ in vitro
Ð¼Ð ÐÐ-Ð²Ð°ÐºÑÐ¸Ð½Ð°
Ð¼Ð ÐÐ-ÑÐµÑÐ°Ð¿Ð¸Ñ
ÑÐ¸Ð½ÑÐµÑÐ¸ÑÐµÑÐºÐ°Ñ Ð¼Ð ÐÐ
Ð ÐÐ-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð° Ð¢7
Ð¿Ð¾Ð»Ð¸Ð°Ð´ÐµÐ½Ð¸Ð»Ð°Ñ-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð°
ÐºÑÐ¿Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸Ðµ
Ð¿Ð¾Ð»Ð¸Ð°Ð´ÐµÐ½Ð¸Ð»Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸Ðµ
Ð¿Ð¾Ð»Ð¸(Ð)-Ñ Ð²Ð¾ÑÑ
Ð¼Ð¾Ð´Ð¸ÑÐ¸ÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð½ÑÐµ Ð½ÑÐºÐ»ÐµÐ¾ÑÐ¸Ð´Ñ
in vitro transcription
mRNA vaccine
mRNA therapy
synthetic mRNA
T7 RNA polymerase
polyadenylate polymerase
capping
polyadenylation
poly(A)-tail
modified nucleotides

description: ÐÐ°Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¿Ð¾ÑÐ²ÑÑÐµÐ½Ð° Ð¾Ð¿ÑÐ¸Ð¼Ð¸Ð·Ð°ÑÐ¸Ð¸ ÑÑÐ»Ð¾Ð²Ð¸Ð¹ ÑÐµÐ°ÐºÑÐ¸Ð¸ ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ð¸ in vitro (IVT). ÐÐ»Ñ Ð¿ÑÐ¾Ð²ÐµÐ´ÐµÐ½Ð¸Ñ ÑÐµÐ°ÐºÑÐ¸Ð¸ Ð¸ Ð¿Ð¾Ð»ÑÑÐµÐ½Ð¸Ñ ÑÑÐ°Ð±Ð¸Ð»ÑÐ½Ð¾Ð¹ Ð¸ ÑÑÐ½ÐºÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½Ð¾Ð¹ Ð¼Ð ÐÐ, ÐºÐ¾Ð´Ð¸ÑÑÑÑÐµÐ¹ ÑÐµÐ¿Ð¾ÑÑÐµÑÐ½ÑÐ¹ Ð³ÐµÐ½ eGFP, Ð² ÑÐ¸ÑÑÐµÐ¼Ðµ ÑÐºÑÐ¿ÑÐµÑÑÐ¸Ð¸ E. coli Ð±ÑÐ»Ð¸ Ð¿Ð¾Ð»ÑÑÐµÐ½Ñ ÑÐµÐºÐ¾Ð¼Ð±Ð¸Ð½Ð°Ð½ÑÐ½ÑÐµ ÑÐµÑÐ¼ÐµÐ½ÑÑ Ð¢7 Ð ÐÐ-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð° Ð¸ Ð¿Ð¾Ð»Ð¸Ð°Ð´ÐµÐ½Ð¸Ð»Ð°Ñ-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð° Ð¸ Ð´Ð¾ÐºÐ°Ð·Ð°Ð½Ð° Ð¸Ñ ÑÑÐ½ÐºÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½Ð¾ÑÑÑ. Ð ÐµÐ°ÐºÑÐ¸Ñ IVT Ð¾Ð¿ÑÐ¸Ð¼Ð¸Ð·Ð¸ÑÐ¾Ð²Ð°Ð»Ð¸ Ð¿Ð¾ ÐºÐ¾Ð»Ð¸ÑÐµÑÑÐ²Ñ Ð¸Ð¾Ð½Ð¾Ð² Ð¼Ð°Ð³Ð½Ð¸Ñ Ð¸ ÑÐ¾Ð¾ÑÐ½Ð¾ÑÐµÐ½Ð¸Ñ ÐºÐ¾Ð½ÑÐµÐ½ÑÑÐ°ÑÐ¸Ð¹ 5-ÐºÑÐ¿-Ð°Ð½Ð°Ð»Ð¾Ð³Ð° Ð¸ Ð³ÑÐ°Ð½Ð¾Ð·Ð¸Ð½ÑÑÐ¸ÑÐ¾ÑÑÐ°ÑÐ°. ÐÑÐ»Ð¸ Ð¿Ð¾Ð»ÑÑÐµÐ½Ñ Ð´Ð²Ð° Ð¾Ð±ÑÐ°Ð·ÑÐ° ÑÐ¸Ð½ÑÐµÑÐ¸ÑÐµÑÐºÐ¾Ð¹ Ð¼Ð ÐÐ, ÑÐ¾Ð´ÐµÑÐ¶Ð°ÑÐ¸Ðµ Ð¼Ð¾Ð´Ð¸ÑÐ¸ÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð½ÑÐµ Ð½ÑÐºÐ»ÐµÐ¾ÑÐ¸Ð´Ñ Ð¿ÑÐµÐ²Ð´Ð¾-UTP Ð¸ 5-Me-CTP Ð¸ ÑÐ¾Ð´ÐµÑÐ¶Ð°ÑÐ¸Ðµ ÐºÐ°Ð½Ð¾Ð½Ð¸ÑÐµÑÐºÐ¸Ðµ Ð½ÑÐºÐ»ÐµÐ¾ÑÐ¸Ð´Ñ. ÐÐ±Ð° Ð²Ð°ÑÐ¸Ð°Ð½ÑÐ° Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½Ð¾Ð¹ Ð¼Ð ÐÐ Ð±ÑÐ»Ð¸ Ð¸Ð½ÐºÐ°Ð¿ÑÑÐ»Ð¸ÑÐ¾Ð²Ð°Ð½Ñ Ð² Ð»Ð¸Ð¿Ð¸Ð´Ð½ÑÐµ Ð½Ð°Ð½Ð¾ÑÐ°ÑÑÐ¸ÑÑ Ð¸ ÑÑÐ°Ð½ÑÑÐ¸ÑÐ¸ÑÐ¾Ð²Ð°Ð½Ñ Ð² ÐºÑÐ»ÑÑÑÑÑ ÑÑÐºÐ°ÑÐ¸Ð¾ÑÐ¸ÑÐµÑÐºÐ¸Ñ ÐºÐ»ÐµÑÐ¾Ðº Ð549. ÐÐ½Ð°Ð»Ð¸Ð· ÑÑÐ°Ð½ÑÐ»ÑÑÐ¸Ð¸ Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½ÑÑ Ð¾Ð±ÑÐ°Ð·ÑÐ¾Ð² Ð¿ÑÐ¾Ð²Ð¾Ð´Ð¸Ð»ÑÑ Ð² ÑÑÐ°Ð²Ð½ÐµÐ½Ð¸Ð¸ Ñ ÐºÐ¾Ð½ÑÑÐ¾Ð»ÑÐ½Ð¾Ð¹ Ð¼Ð ÐÐ Ð¸ Ð¿ÑÐ¾Ð²Ð¾Ð´Ð¸Ð»ÑÑ ÑÑÐ°ÑÐ¸ÑÑÐ¸ÑÐµÑÐºÐ¸Ð¹ Ð°Ð½Ð°Ð»Ð¸Ð· ÑÑÐµÐ´Ð½Ð¸Ñ Ð·Ð½Ð°ÑÐµÐ½Ð¸Ð¹ ÑÑÐ°Ð½ÑÑÐµÐºÑÐ¸Ð¸ Ð¸ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸. ÐÑÑÐ¾ÐºÐ¸Ð¹ ÑÑÐ¾Ð²ÐµÐ½Ñ ÑÑÐ°Ð½ÑÑÐµÐºÑÐ¸Ð¸ Ð±ÑÐ» Ð¾Ð±Ð½Ð°ÑÑÐ¶ÐµÐ½ Ð² Ð¼Ð ÐÐ Ñ Ð¼Ð¾Ð´Ð¸ÑÐ¸ÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð½ÑÐ¼Ð¸ Ð½ÑÐºÐ»ÐµÐ¾ÑÐ¸Ð´Ð°Ð¼Ð¸, Ð² Ð¾ÑÐ»Ð¸ÑÐ¸Ðµ Ð¾Ñ Ð¾Ð±ÑÐ°Ð·ÑÐ° Ñ Ð½ÐµÐ¼Ð¾Ð´Ð¸ÑÐ¸ÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð½ÑÐ¼Ð¸ Ð½ÑÐºÐ»ÐµÐ¾ÑÐ¸Ð´Ð°Ð¼Ð¸, ÐºÐ¾ÑÐ¾ÑÑÐ¹ Ð¿Ð¾ÐºÐ°Ð·Ð°Ð» ÑÑÑÐµÑÑÐ²ÐµÐ½Ð½Ð¾ Ð½Ð¸Ð·ÐºÐ¸Ð¹ ÑÑÐ¾Ð²ÐµÐ½Ñ. Ð£ÑÐ¾Ð²ÐµÐ½Ñ ÑÑÐ°Ð½ÑÐ»ÑÑÐ¸Ð¸ Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½ÑÑ Ð¼Ð ÐÐ Ð±ÑÐ» Ð·Ð½Ð°ÑÐ¸ÑÐµÐ»ÑÐ½Ð¾ Ð½Ð¸Ð¶Ðµ, ÑÐµÐ¼ Ñ ÐºÐ¾Ð½ÑÑÐ¾Ð»ÑÐ½Ð¾Ð³Ð¾ Ð¾Ð±ÑÐ°Ð·ÑÐ°, ÑÑÐ¾ Ð¼Ð¾Ð¶ÐµÑ Ð±ÑÑÑ ÑÐ²ÑÐ·Ð°Ð½Ð¾ Ñ Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½Ð¸ÐµÐ¼ Ð´ÑÑÐ³Ð¾Ð³Ð¾ ÑÐ¸Ð¿Ð° Ð°Ð½Ð°Ð»Ð¾Ð³Ð° ÐºÑÐ¿Ð° Ð¸ Ð½ÐµÐ¾Ð±ÑÐ¾Ð´Ð¸Ð¼Ð¾ÑÑÑÑ Ð¾Ð¿ÑÐ¸Ð¼Ð¸Ð·Ð°ÑÐ¸Ð¸ ÑÑÐ»Ð¾Ð²Ð¸Ð¹ ÑÐµÐ°ÐºÑÐ¸Ð¸ Ð¿Ð¾Ð»Ð¸Ð°Ð´ÐµÐ½Ð¸Ð»Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸Ñ. Ð ÐµÐ·ÑÐ»ÑÑÐ°ÑÑ Ð¿Ð¾ÐºÐ°Ð·Ð°Ð»Ð¸, ÑÑÐ¾ Ð¾Ð±Ð° Ð¾Ð±ÑÐ°Ð·ÑÐ° Ð¼Ð ÐÐ, Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½ÑÐµ Ñ Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½Ð¸ÐµÐ¼ Ð¾ÑÐ¸Ð³Ð¸Ð½Ð°Ð»ÑÐ½ÑÑ ÑÐµÐºÐ¾Ð¼Ð±Ð¸Ð½Ð°Ð½ÑÐ½ÑÑ ÑÐµÑÐ¼ÐµÐ½ÑÐ¾Ð² Ð¢7 Ð ÐÐ-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ñ Ð¸ Ð¿Ð¾Ð»Ð¸Ð°Ð´ÐµÐ½Ð¸Ð»Ð°Ñ-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ñ, ÑÑÐ½ÐºÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½Ñ Ð¸ ÑÐ¿Ð¾ÑÐ¾Ð±Ð½Ñ ÑÑÐ°Ð½ÑÐ»Ð¸ÑÐ¾Ð²Ð°ÑÑ ÐºÐ¾Ð´Ð¸ÑÑÐµÐ¼ÑÐ¹ Ð±ÐµÐ»Ð¾Ðº.
description: The given work is devoted to the optimization of the reaction conditions for in vitro transcription (IVT). To carry out the reaction and obtain a stable and functional mRNA encoding the eGFP reporter gene, recombinant enzymes T7 RNA polymerase and polyadenylate polymerase were obtained in the E. coli expression system, and their functionality was proved. The IVT reaction was optimized for the quantity of magnesium ions and the ratio of the concentrations of the 5 cap analog and guanosine triphosphate. Two synthetic mRNA samples were obtained containing modified pseudo-UTP and 5-Me-CTP nucleotides and containing canonical nucleotides. Both variants of the resulting mRNA were encapsulated in lipid nanoparticles and transfected into a culture of A549 eukaryotic cells. The analysis of the translation of the obtained samples was carried out in comparison with the control mRNA and a statistical analysis of the mean values of transfection and fluorescence was performed. A high level of transfection was found in mRNA with modified nucleotides, in contrast to the sample with unmodified nucleotides, which showed a significantly low level. The level of translation of the obtained mRNAs was significantly lower than that of the control sample, which may be due to the use of a different type of cap analogue and the need to optimize the conditions of the polyadenylation reaction. The results showed that both mRNA samples obtained using the original recombinant enzymes T7 RNA polymerase and polyadenylate-polymerase are functional and able to translate the encoded protein.