Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities
Gene regulation by trans-acting small RNAs (sRNAs) has considerable advantages over other gene regulation strategies. However, synthetic sRNAs mainly take natural sRNAs (MicC or SgrS) as backbones and comprise three functional elements folding into two or more stem-loop structures: an mRNA base pair...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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KeAi Communications Co., Ltd.
2023-03-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X22001193 |
| _version_ | 1797897201165271040 |
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| author | Yang Wang Guobin Yin Huanjiao Weng Luyao Zhang Guocheng Du Jian Chen Zhen Kang |
| author_facet | Yang Wang Guobin Yin Huanjiao Weng Luyao Zhang Guocheng Du Jian Chen Zhen Kang |
| author_sort | Yang Wang |
| collection | DOAJ |
| description | Gene regulation by trans-acting small RNAs (sRNAs) has considerable advantages over other gene regulation strategies. However, synthetic sRNAs mainly take natural sRNAs (MicC or SgrS) as backbones and comprise three functional elements folding into two or more stem-loop structures: an mRNA base pairing region, an Hfq-binding structure, and a rho-independent terminator. Due to limited numbers of natural sRNAs and complicated backbone structures, synthetic sRNAs suffer from low activity programmability and poor structural modularity. Moreover, natural sRNA backbone sequences may increase the possibility of unwanted recombination. Here, we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs (ssl-sRNAs), which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons. Particularly, ssl-sRNA requires no independent Hfq-binding structure, as the polyU tail fulfills the roles of binding Hfq. A thermodynamic-based scoring model and a web server sslRNAD (http://www.kangzlab.cn/) were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities. ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs. The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis. A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries. As examples, the E. coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs. ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes. |
| first_indexed | 2024-04-10T07:54:53Z |
| format | Article |
| id | doaj.art-048c6685c2554c169a263b7871bfb181 |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-04-10T07:54:53Z |
| publishDate | 2023-03-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-048c6685c2554c169a263b7871bfb1812023-02-23T04:31:43ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2023-03-01818696Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activitiesYang Wang0Guobin Yin1Huanjiao Weng2Luyao Zhang3Guocheng Du4Jian Chen5Zhen Kang6The Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaThe Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, ChinaThe Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, ChinaThe Science Center for Future Foods, Jiangnan University, Wuxi, 214122, ChinaThe Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaThe Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaThe Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China; The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Corresponding author. The Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.Gene regulation by trans-acting small RNAs (sRNAs) has considerable advantages over other gene regulation strategies. However, synthetic sRNAs mainly take natural sRNAs (MicC or SgrS) as backbones and comprise three functional elements folding into two or more stem-loop structures: an mRNA base pairing region, an Hfq-binding structure, and a rho-independent terminator. Due to limited numbers of natural sRNAs and complicated backbone structures, synthetic sRNAs suffer from low activity programmability and poor structural modularity. Moreover, natural sRNA backbone sequences may increase the possibility of unwanted recombination. Here, we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs (ssl-sRNAs), which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons. Particularly, ssl-sRNA requires no independent Hfq-binding structure, as the polyU tail fulfills the roles of binding Hfq. A thermodynamic-based scoring model and a web server sslRNAD (http://www.kangzlab.cn/) were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities. ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs. The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis. A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries. As examples, the E. coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs. ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.http://www.sciencedirect.com/science/article/pii/S2405805X22001193Regulatory RNAMetabolic engineeringSynthetic biologyDe novo designErgothioneine |
| spellingShingle | Yang Wang Guobin Yin Huanjiao Weng Luyao Zhang Guocheng Du Jian Chen Zhen Kang Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities Synthetic and Systems Biotechnology Regulatory RNA Metabolic engineering Synthetic biology De novo design Ergothioneine |
| title | Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities |
| title_full | Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities |
| title_fullStr | Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities |
| title_full_unstemmed | Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities |
| title_short | Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities |
| title_sort | gene knockdown by structure defined single stem loop small non coding rnas with programmable regulatory activities |
| topic | Regulatory RNA Metabolic engineering Synthetic biology De novo design Ergothioneine |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X22001193 |
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