Kim Yoon-Seob, Kim Do-Hyung, An Daegi, Lim Younghyun, Seo Young-Jin, Kim Hak Kyun, Kang Ho-Young
Drug Discovery Center, NuclixBio, Seoul, Republic of Korea.
Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
Anim Cells Syst (Seoul). 2023 Oct 4;27(1):208-218. doi: 10.1080/19768354.2023.2265165. eCollection 2023.
Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.
环状RNA（circRNA）是一种具有共价闭合环状结构的非编码RNA，通常比信使RNA（mRNA）更稳定。然而，circRNA中位于内部核糖体进入位点（IRES）之后的编码序列（CDS）可以被翻译，并且这一特性最近已被用于生产蛋白质作为新型治疗工具。然而，由于长链RNA的环化效率低，从circRNA中生产大蛋白很困难。在本研究中，我们报告了我们使用RNA连接酶1和夹板DNA成功地通过夹板DNA连接法合成了circRNA，夹板DNA包含与前体线性RNA两端互补的序列。与不使用夹板DNA的传统酶法相比，该方法能实现更高效的环化，能轻松产生表达相对较大蛋白（包括IgG重链和轻链）的circRNA。更长的夹板DNA（42个核苷酸）在环化中更有效。此外，使用含有腺嘌呤类似物2,6-二氨基嘌呤（DAP）的夹板DNA，可能通过加强夹板DNA与前体RNA之间的相互作用来提高环化效率。夹板DNA连接法高效生产circRNA所需的夹板DNA比前体RNA多5倍，但我们改进的夹板DNA连接法可以使用与前体RNA等量的夹板DNA来生产circRNA。我们改进的夹板DNA连接法将有助于开发利用circRNA的新型治疗工具，用于治疗各种疾病以及开发人用和兽用疫苗。
