مهندسي RNA حلقوي به منظور ترجمه قوي و پايدار در سلول‌هاي يوكاريوتي

Engineering circular RNA for potent and stable translation in eukaryotic cells

RNA ﭘﻴﻚ )mRNA( ﭘﺘﺎﻧﺴﻴﻞ ﮔﺴﺘﺮده اي ﺑﺮاي اﺳﺘﻔﺎده در ﺳﻴﺴﺘﻢ ﻫﺎي ﺑﻴﻮﻟﻮژﻳﻜﻲ دارد. ﺑﺎ اﻳﻦ ﺣﺎل، ﻳﻚ ﻣﺤﺪودﻳﺖ اﺳﺎﺳﻲ در اﺳﺘﻔﺎده از آن، ﻧﻴﻤﻪ ﻋﻤﺮ ﻧﺴﺒﺘﺎً ﻛﻮﺗﺎه آن در اﻳﻦ ﺳﻴﺴﺘﻢ ﻫﺎ اﺳﺖ. در اﻳﻨﺠﺎ ﻣﺎ RNA ﺣﻠﻘﻮي )circRNA( اﮔﺰوژﻧﻲ ﺑﺎ ﻣﻨﺸﺎ ﺧﺎرﺟﻲ( را ﺗﻮﺳﻌﻪ دادﻳﻢ ﺗﺎ ﻣﺪت زﻣﺎن ﺑﻴﺎن ﭘﺮوﺗﺌﻴﻦ از RNA ﻳﻲ ﺑﺎ ﻃﻮل ﻛﺎﻣﻞ را، اﻓﺰاﻳﺶ دﻫﻴﻢ. ﻧﺨﺴﺖ، ﻣﺎ ﻳﻚ اﻳﻨﺘﺮون ﺧﻮد ﭘﻴﺮاﻳﺸﮕﺮ )self-splicing intron( را ﻃﻮري ﻃﺮاﺣﻲ ﻛﺮدﻳﻢ ﺗﺎ ﺑﻪ وﺳﻴﻠﻪ ﻳﻚ ﻃﺮاﺣﻲ ﻣﻨﻄﻘﻲ )rationally designing( ﺑﻪ ﻛﻤﻚ ﺗﻮاﻟﻲ ﻫﺎي ﻣﻮﺟﻮد ﻛﻪ ﺑﻪ ﻧﺤﻮ ﻣﻮﺛﺮي اﺳﭙﻼﻳﺴﻴﻨﮓ ﻛﻤﻚ ﻣﻲ ﻛﻨﻨﺪ، در ﺷﺮاﻳﻂ آزﻣﺎﻳﺸﮕﺎﻫﻲ )in vitro(، داﻣﻨﻪ ﮔﺴﺘﺮده اي از ﺗﻮاﻟﻲ ﻫﺎي ﺑﺎ ﻃﻮل ﺗﺎ 5kb را، ﺣﻠﻘﻮي ﺳﺎزد. در اداﻣﻪ ﺗﺮﺟﻤﻪ ﭘﺮوﺗﺌﻴﻦ ﻛﺎرﺑﺮدي از اﻳﻦ circRNAﻫﺎ را در ﺳﻠﻮل ﻫﺎي ﻳﻮﻛﺎرﻳﻮﺗﻴﻚ ﺑﻪ ﺣﺪاﻛﺜﺮ ﻣﻲ رﺳﺎﻧﻴﻢ و در ﺧﻮاﻫﻴﻢ ﻳﺎﻓﺖ ﻛﻪ circRNAﻫﺎي ﻣﻬﻨﺪﺳﻲ ﺷﺪه ﻛﻪ ﺑﻪ وﺳﻴﻠﻪ ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﻣﺎﻳﻊ ﺑﺎ ﻋﻤﻠﻜﺮد ﺑﺎﻻ )high performance liquid chromatography( ﺗﺨﻠﻴﺺ ﺷﺪه اﻧﺪ ﻛﻴﻔﻴﺖ ﺑﺎﻻي ﺗﻮﻟﻴﺪ ﭘﺮوﺗﺌﻴﻦ را از ﻧﻈﺮ ﻣﻘﺪار ﭘﺮوﺗﺌﻴﻦ ﺗﻮﻟﻴﺪ ﺷﺪه وﻫﻤﭽﻨﻴﻦ ﭘﺎﻳﺪاري ﺗﻮﻟﻴﺪ ﭘﺮوﺗﺌﻴﻦ، ﻓﺮاﻫﻢ ﻣﻲ ﻛﻨﻨﺪ. اﻳﻦ ﻳﻜﻲ اوﻟﻴﻦ ﻣﻄﺎﻟﻌﺎﺗﻲ اﺳﺖ ﻛﻪ از circRNA اﮔﺰوژن ﺑﺮاي ﺑﻴﺎن ﻗﻮي و ﭘﺎﻳﺪار ﭘﺮوﺗﺌﻴﻦ در ﺳﻠﻮﻟﻬﺎي ﻳﻮﻛﺎرﻳﻮﺗﻲ اﺳﺘﻔﺎده ﻛﺮده اﺳﺖ و ﻧﺸﺎن داده ﻛﻪ circRNA، ﺟﺎﻳﮕﺰﻳﻦ ﻣﻨﺎﺳﺐ ﺑﺮاي mRNA ﻫﺎي ﺧﻄﻲ ﻣﻲ ﺑﺎﺷﺪ.

Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.