Characterization of group I introns in generating circular RNAs as vaccines. Characterization of group I introns in generating circular RNAs as vaccines

Group 1 introns can be utilized for efficient circularization of RNA molecules. However different group 1 introns are known to have different properties, including having different efficiencies of circularization and generating different levels of immune response inside cells (reactogenicity). Here, we investigated generating circular RNAs using permuted intron-exon (PIE) systems of group 1 introns from different organisms. We observed that group 1 intron sequences have variable circularization efficiency that is size dependent and circularize with high accuracy. We also determined the sequence properties of the exon1 and exon2 domains of different group1 introns and showed that we can engineer elements such as S1 tag into this domain for better purification. Structural probing and mutational analysis in group 1 introns and their PIE RNAs showed that structural features in P2 and P9 domains are correlated with circularization efficiency and that swapping sequences can improve circularization efficiency. Lastly, we showed that circular RNAs made by Sd group 1 introns elicit lower amount of reactogenicity and either An or Sd can act as effective RNA vaccines. Our work deepens the understanding of the properties of group 1 introns and their use in circular RNA production and medicine. Overall design: To investigate whether different group 1 introns can enable RNA circularization upon PIE re-arrangement, we tested 8 group 1 intron sequences across a range of different buffers, differing in salt and temperature to identify the condition for efficient circularization (Supp Table 1). These 8 group 1 introns are from T4 phage (T4), Anabaena (An), Scytalidium dimidiatum (Sd), Clostridium botulinum (Cb), Scytonema hofmanni (Sh), Geosmithia virida (Gv), Penicillium oblatum (Po), and Barrmaelia oxyacanthae (Bo) (15-18). PIE constructs were made by splitting these group 1 introns at the P6 region and moving 3’-intron and exon 2 (3’I-E2) sequences to the 5’ end and exon 1 and 5’-intron (E1-5’I) sequences to the 3’ end (Figure 1A, B). For initial testing purpose, a 0.5kb Gaussia luciferase (Gluc) gene is used as an insert between 3’I-E2 and E1-5’I. To test circularization, all T4, An, Sd, Cb, Sh, Gv, Po, Bo PIE Gluc RNAs were in-vitro transcribed using T7 RNA polymerase and were incubated in different buffer conditions at different temperatures for circularization (A to G, Methods). To determine of exon sequence requirement for circularization, T4 PIE N60 Gluc, An PIE N60 Gluc, Sh PIE N60 Gluc, and Po PIE N60 Gluc IVT RNA were folded in condition B for 15 minutes or 1 hour. These samples were resolved by agarose gel and the lower band in each sample containing circular RNA was gel extracted for sequencing To analyze the circular RNA formation accuracy, synthesis of first-stand cDNA from circular Gluc RNA was performed using SuperScript III reverse transcriptase (Thermo Fisher) with primer junction R according to manufacturer’s instructions. Subsequently, this cDNA was used as template for PCR amplification of 6 junction fragments with respective T4, An, Sd, Cb, Sh, and Po junction F and R primers. Each junction PCR fragment was processed using NEBNext Ultra II DNA Library Prep Kit to construct libraries compatible for Illumina sequencing. To investigate sequences of full circular RNA, same cDNA template was used for full circular PCR amplification with respective T4, An, Sd, Cb, Sh and Po full circle F and R primers. Then these full circle fragments were sequenced using Nanopore ligation sequencing kit.

Group I内含子（Group 1 introns）可用于高效实现RNA分子的环化。然而不同的Group I内含子具有各异的特性，包括存在差异的环化效率，以及在细胞内引发不同水平的免疫应答（反应原性，reactogenicity）。本研究针对利用不同生物来源的Group I内含子的剪接内含子外显子重排（permuted intron-exon, PIE）系统制备环状RNA的方法展开探究。研究结果显示，Group I内含子序列的环化效率存在差异，且该差异具有尺寸依赖性，同时环化过程具备较高的准确性。此外，本研究还解析了不同Group I内含子的外显子1（exon1）与外显子2（exon2）结构域的序列特性，并证实可在该结构域中插入诸如S1标签（S1 tag）等元件以实现更高效的纯化。对Group I内含子及其PIE RNA的结构探测与突变分析表明，P2与P9结构域的结构特征与环化效率密切相关，且序列互换可有效提升环化效率。最后，本研究证实，由Sd来源的Group I内含子制备的环状RNA引发的反应原性更低，且An或Sd来源的环状RNA均可作为有效的RNA疫苗。本研究深化了人们对Group I内含子特性及其在环状RNA制备与医学应用中应用的理解。整体实验设计：为探究不同Group I内含子能否在PIE重排后实现RNA环化，本研究测试了8种不同来源的Group I内含子序列，并在一系列盐浓度与温度参数各异的缓冲液中开展实验，以筛选高效环化的最优条件（补充表1）。这8种Group I内含子分别来自T4噬菌体（T4）、鱼腥藻（Anabaena, An）、双态帚枝霉（Scytalidium dimidiatum, Sd）、肉毒梭菌（Clostridium botulinum, Cb）、霍夫曼氏鞘丝藻（Scytonema hofmanni, Sh）、绿形地丝霉（Geosmithia virida, Gv）、宽形青霉（Penicillium oblatum, Po）以及山楂巴雷霉（Barrmaelia oxyacanthae, Bo）(15-18)。PIE构建体的制备方式为：将这些Group I内含子在P6区域处切开，将3'端内含子与外显子2（3'I-E2）序列移至5'端，同时将外显子1与5'端内含子（E1-5'I）序列移至3'端（图1A、B）。为进行初步测试，本研究在3'I-E2与E1-5'I之间插入了一段0.5kb的高斯荧光素酶（Gaussia luciferase, Gluc）基因作为报告插入片段。为验证环化效果，我们利用T7 RNA聚合酶（T7 RNA polymerase）体外转录获得所有T4、An、Sd、Cb、Sh、Gv、Po、Bo的PIE-Gluc RNA，并将其置于不同缓冲液条件与温度下孵育以进行环化反应（A至G组，详见方法部分）。为明确环化所需的外显子序列特征，我们将T4 PIE N60 Gluc、An PIE N60 Gluc、Sh PIE N60 Gluc以及Po PIE N60 Gluc的体外转录RNA在B条件下分别孵育15分钟与1小时。将这些样品通过琼脂糖凝胶电泳分离，切取每个样品中包含环状RNA的低分子量条带进行胶回收并测序。为分析环状RNA的形成准确性，我们根据制造商说明书，使用SuperScript III反转录酶（SuperScript III reverse transcriptase, Thermo Fisher）与引物Junction R，从环状Gluc RNA中合成第一链cDNA。随后以该cDNA为模板，使用对应T4、An、Sd、Cb、Sh及Po的junction F与R引物，通过PCR扩增6个连接片段。每个连接PCR产物均使用NEBNext Ultra II DNA文库制备试剂盒（NEBNext Ultra II DNA Library Prep Kit）进行处理，以构建适配Illumina测序的文库。为解析完整环状RNA的序列，我们使用相同的cDNA模板，通过对应T4、An、Sd、Cb、Sh及Po的全环F与R引物进行全环状RNA的PCR扩增。随后使用Nanopore连接测序试剂盒（Nanopore ligation sequencing kit）对这些全环扩增片段进行测序。