The synthesis of Broccoli RNA fluorescent aptamer in Saccharomyces cerevisiae yeast cells

Abstract

BACKGROUND: RNA aptamers are short, single-stranded oligonucleotides, with remarkable binding ability to target molecules characterized by high specificity and affinity. Such targets are vastly diverse and range from specific ions to entire cells. RNA aptamers are widely used in biology and medicine for basic research, as well as for practical purposes as in therapy and diagnostics. At present, chemical or in vitro methods of synthesis are mainly used to obtain RNA aptamers. However, such methods are expensive and time-consuming with low productivity. Therefore, in vivo methods are becoming more attractive to researchers working on optimizing high-scale production of RNA aptamers.

AIM: The aim of this work is to develop a reporter system for optimizing the synthesis of small RNA molecules in Saccharomyces cerevisiae yeast cells.

MATERIALS AND METHODS: We used the Broccoli fluorescent RNA aptamer to develop a reporter system allowing us to optimize the conditions for in vivo short RNA synthesis in yeast cells. This aptamer is about 112 bp in size and binds to the fluorogenic dye DFHBI-1T. Only upon binding, the aptamer-dye complex exhibits fluorescence properties. After excitation using light with a wavelength of 482 nm, the aptamer-dye complex emission is observed with a peak at 505 nm.

RESULTS: We have designed a reporter system providing the synthesis of the fluorescent Broccoli RNA aptamer in S. cerevisiae yeast cells. Transcription of RNA molecules containing the aptamer is carried out by the regulated promoter of the GAL1 gene. The synthesized transcripts contain the HH and HDV ribozymes to ensure precise cleavage of the RNA aptamer sequences.

CONCLUSIONS: This reporter system is based on the Broccoli RNA aptamer, and it can be used to optimize the in vivo synthesis of RNA aptamers in S. cerevisiae yeast cells. This work serves an urgent task in connection with the active use of such aptamers in scientific research, biotechnology and medicine.

Keywords

yeast / Saccharomyces cerevisiae / reporter systems / fluorescent RNA aptamers

References

[1] Barnett JA. A history of research on yeasts 10: foundations of yeast genetics. Yeast. 2007;24(10):799–845. DOI: 10.1002/yea.1513

[2] Barnett J.A. A history of research on yeasts 10: foundations of yeast genetics // Yeast. 2007. Vol. 24, No. 10. P. 799–845. DOI: 10.1002/yea.1513

[3] Botstein D, Fink GR. Yeast: an experimental organism for 21st Century biology. Genetics. 2011;189(3):695–704. DOI: 10.1534/genetics.111.130765

[4] Botstein D., Fink G.R. Yeast: an experimental organism for 21st Century biology // Genetics. 2011. Vol. 189, No. 3. P. 695–704. DOI: 10.1534/genetics.111.130765

[5] Goffeau A, Barrell BG, Bussey H, et al. Life with 6000 Genes. Science. 1996;274(5287):546–567. DOI: 10.1126/science.274.5287.546

[6] Goffeau A., Barrell B.G., Bussey H., et al. Life with 6000 Genes // Science. 1996. Vol. 274, No. 5287. P. 546–567. DOI: 10.1126/science.274.5287.546

[7] Karathia H, Vilaprinyo E, Sorribas A, Alves R. Saccharomyces cerevisiae as a model organism: a comparative study. PLoS One. 2011;6(2): e16015. DOI: 10.1371/journal.pone.0016015

[8] Karathia H., Vilaprinyo E., Sorribas A., Alves R. Saccharomyces cerevisiae as a model organism: a comparative study // PLoS One. 2011. Vol. 6, No. 2. ID e16015. DOI: 10.1371/journal.pone.0016015

[9] Bolotin-Fukuhara M, Dumas B, Gaillardin C. Yeasts as a model for human diseases. FEMS Yeast Research. 2010;10(8):959–960. DOI: 10.1111/j.1567-1364.2010.00693.x

[10] Bolotin-Fukuhara M., Dumas B., Gaillardin C. Yeasts as a model for human diseases // FEMS Yeast Research. 2010. Vol. 10, No. 8. P. 959–960. DOI: 10.1111/j.1567-1364.2010.00693.x

[11] Guthrie C, Fink GR. Guide to Yeast Genetics and Molecular Biology. Academic Press, Cambridge, 1991. 194 p.

[12] Guthrie C., Fink G.R. Guide to yeast genetics and molecular biology. Academic Press, Cambridge, 1991. 194 p.

[13] Pronk JT. Auxotrophic yeast strains in fundamental and applied research. Appl Environ Microbiol. 2002;68(5):2095–2100. DOI: 10.1128/AEM.68.5.2095-2100.2002

[14] Pronk J.T. Auxotrophic yeast strains in fundamental and applied research // Appl Environ Microbiol. 2002. Vol. 68, No. 5. P. 2095–2100. DOI: 10.1128/AEM.68.5.2095-2100.2002

[15] Lohr D, Venkov P, Zlatanova J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network. FASEB J. 1995;9(9):777–787. DOI: 10.1096/fasebj.9.9.7601342

[16] Lohr D., Venkov P., Zlatanova J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network // FASEB J. 1995. Vol. 9, No. 9. P. 777–787. DOI: 10.1096/fasebj.9.9.7601342

[17] Nielsen J. Yeast systems biology: model organism and cell factory. Biotechnol J. 2019;14(9): e1800421. DOI: 10.1002/biot.201800421

[18] Nielsen J. Yeast systems biology: model organism and cell factory // Biotechnol J. 2019. Vol. 14, No. 9. ID e1800421. DOI: 10.1002/biot.201800421

[19] Macreadie I, Dhakal S. “The awesome power of yeast”. Microbiology Australia. 2022;43(1):19–21. DOI: 10.1071/ma22007

[20] Macreadie I., Dhakal S. “The awesome power of yeast” // Microbiology Australia. 2022. Vol. 43, No. 1. P. 19–21. DOI: 10.1071/ma22007

[21] Szczebara FM, Chandelier C, Villeret C, et al. Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol. 2003;21(2):143–149. DOI: 10.1038/nbt775

[22] Szczebara F.M., Chandelier C., Villeret C., et al. Total biosynthesis of hydrocortisone from a simple carbon source in yeast // Nat Biotechnol. 2003. Vol. 21, No. 2. P. 143–149. DOI: 10.1038/nbt775

[23] Paddon CJ, Westfall PJ, Pitera DJ, et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013;496(7446):528–532. DOI: 10.1038/nature12051

[24] Paddon C.J., Westfall P.J., Pitera D.J., et al. High-level semi-synthetic production of the potent antimalarial artemisinin // Nature. 2013. Vol. 496, No. 7446. P. 528–532. DOI: 10.1038/nature12051

[25] Klussmann S. The Aptamer Handbook: Functional Oligonucleotides and Their Applications // John Wiley & Sons. 2006. 500 p. DOI: 10.1002/3527608192

[26] Klussmann S. The Aptamer Handbook: Functional Oligonucleotides And Their Applications // John Wiley & Sons. 2006. 500 p. DOI: 10.1002/3527608192

[27] Lee JF. Aptamer Database. Nucleic Acids Research. 2004;32(S1):95D–D100. DOI: 10.1093/nar/gkh094

[28] Lee J.F. Aptamer Database // Nucleic Acids Research. 2004. Vol. 32, No. S1. P. 95D–D100. DOI: 10.1093/nar/gkh094

[29] Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346:818–822. DOI: 10.1038/346818a0

[30] Ellington A.D., Szostak J.W. In vitro selection of RNA molecules that bind specific ligands // Nature. 1990. Vol. 346. P. 818–822. DOI: 10.1038/346818a0

[31] Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249(4968):505–510. DOI: 10.1126/science.2200121

[32] Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase // Science. 1990. Vol. 249, No. 4968. P. 505–510. DOI: 10.1126/science.2200121

[33] Filonov GS, Moon JD, Svensen N, Jaffrey SR. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J Am Chem Soc. 2014;136(46):16299–16308. DOI: 10.1021/ja508478x

[34] Filonov G.S., Moon J.D., Svensen N., Jaffrey S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution // J Am Chem Soc. 2014. Vol. 136, No. 46. P. 16299–16308. DOI: 10.1021/ja508478x

[35] Paige JS, Wu KY, Jaffrey SR. RNA mimics of green fluorescent protein. Science. 2011;333(6042):642–646. DOI: 10.1126/science.1207339

[36] Paige J.S., Wu K.Y., Jaffrey S.R. RNA Mimics of Green Fluorescent Protein // Science. 2011. Vol. 333, No. 6042. P. 642–646. DOI: 10.1126/science.1207339

[37] Filonov GS, Kam CW, Song W, Jaffrey SR. In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies. Chem Biol. 2015;22(5):649–660. DOI: 10.1016/j.chembiol.2015.04.018

[38] Filonov G.S., Kam C.W., Song W., Jaffrey S.R. In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies // Chem Biol. 2015. Vol. 22, No. 5. P. 649–660. DOI: 10.1016/j.chembiol.2015.04.018

[39] Song W, Strack RL, Jaffrey SR. Imaging bacterial protein expression using genetically encoded RNA sensors. Nat Methods. 2013;10(9):873–875. DOI: 10.1038/nmeth.2568

[40] Song W., Strack R.L., Jaffrey S.R. Imaging bacterial protein expression using genetically encoded RNA sensors // Nat Methods. 2013. Vol. 10, No. 9. P. 873–875. DOI: 10.1038/nmeth.2568

[41] McConnell EM, Nguyen J, Li Y. Aptamer-based biosensors for environmental monitoring. Front Chem. 2020;8:434. DOI: 10.3389/fchem.2020.00434

[42] McConnell E.M., Nguyen J., Li Y. Aptamer-based biosensors for environmental monitoring // Front Chem. 2020. Vol. 8. ID434. DOI: 10.3389/fchem.2020.00434

[43] Wiedman GR, Zhao Y, Mustaev A, et al. An aptamer-based biosensor for the azole class of antifungal drugs. mSphere. 2017;2(4): e00274–17. DOI: 10.1128/mSphere.00274-17.

[44] Wiedman G.R., Zhao Y., Mustaev A., et al. An Aptamer-based biosensor for the azole class of antifungal drugs // mSphere. 2017. Vol. 2, No. 4. ID e00274–17. DOI: 10.1128/mSphere.00274-17.

[45] Wu S, Zhang H, Shi Z, et al. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles. Food Control. 2015;50:597–604. DOI: 10.1016/j.foodcont.2014.10.003

[46] Wu S., Zhang H., Shi Z., et al. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles // Food Control. 2015. Vol. 50. P. 597–604. DOI: 10.1016/j.foodcont.2014.10.003

[47] Eissa S, Zourob M. In vitro selection of DNA aptamers targeting β-lactoglobulin and their integration in graphene-based biosensor for the detection of milk allergen. Biosens Bioelectron. 2017;91:169–174. DOI: 10.1016/j.bios.2016.12.020

[48] Eissa S., Zourob M. In vitro selection of DNA aptamers targeting β-lactoglobulin and their integration in graphene-based biosensor for the detection of milk allergen // Biosens Bioelectron. 2017. Vol. 91. P. 169–174. DOI: 10.1016/j.bios.2016.12.020

[49] Yang YB, Yang XD, Zou XM, et al. Ultrafine graphene nanomesh with large on/off ratio for high-performance flexible biosensors. Adv Funct Mater. 2017;27(19):1604096. DOI: 10.1002/adfm.201604096

[50] Yang Y.B., Yang X.D., Zou X.M., et al. Ultrafine graphene nanomesh with large on/off ratio for high-performance flexible biosensors // Adv Funct Mater. 2017. Vol. 27, No. 19. ID 1604096. DOI: 10.1002/adfm.201604096

[51] Alizadeh N, Memar MY, Moaddab SR, Kafil HS. Aptamer-assisted novel technologies for detecting bacterial pathogens. Biomed Pharmacother. 2017;93:737–745. DOI: 10.1016/j.biopha.2017.07.011

[52] Alizadeh N., Memar M.Y., Moaddab S.R., Kafil H.S. Aptamer-assisted novel technologies for detecting bacterial pathogens // Biomed Pharmacother. 2017. Vol. 93. P. 737–745. DOI: 10.1016/j.biopha.2017.07.011

[53] Hoffmann S, Hoos J, Klussmann S, Vonhoff S. RNA aptamers and spiegelmers: synthesis, purification, and post-synthetic PEG conjugation. Curr Protoc Nucleic Acid Chem. 2011;4. DOI: 10.1002/0471142700.nc0446s46

[54] Hoffmann S., Hoos J., Klussmann S., Vonhoff S. RNA aptamers and spiegelmers: synthesis, purification, and post-synthetic PEG conjugation // Curr Protoc Nucleic Acid Chem. 2011. Vol. 4. DOI: 10.1002/0471142700.nc0446s46

[55] Kartje ZJ, Janis HI, Mukhopadhyay S, Gagnon KT. Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter. J Biol Chem. 2021;296:100175. DOI: 10.1074/jbc.RA120.014553

[56] Kartje Z.J., Janis H.I., Mukhopadhyay S., Gagnon K.T. Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter // J Biol Chem. 2021. Vol. 296. ID100175. DOI: 10.1074/jbc.RA120.014553

[57] Duman-Scheel M. Saccharomyces cerevisiae (Baker’s Yeast) as an interfering RNA expression and delivery system. Curr Drug Targets. 2019;20(9):942–952. DOI: 10.2174/1389450120666181126123538

[58] Duman-Scheel M. Saccharomyces cerevisiae (Baker’s Yeast) as an interfering RNA expression and delivery system // Curr Drug Targets. 2019. Vol. 20, No. 9. P. 942–952. DOI: 10.2174/1389450120666181126123538

[59] Garrait G, Jarrige JF, Blanquet-Diot S, Alric M. Genetically engineered yeasts as a new delivery vehicle of active compounds to the digestive tract: in vivo validation of the concept in the rat. Metab Eng. 2009;11(3):148–154. DOI: 10.1016/j.ymben.2009.01.001

[60] Garrait G., Jarrige J.F., Blanquet-Diot S., Alric M. Genetically engineered yeasts as a new delivery vehicle of active compounds to the digestive tract: in vivo validation of the concept in the rat // Metab Eng. 2009. Vol. 11, No. 3. P. 148–154. DOI: 10.1016/j.ymben.2009.01.001

[61] Phelps MP, Seeb LW, Seeb JE. Transforming ecology and conservation biology through genome editing. Conserv Biol. 2020;34(1): 54–65. DOI: 10.1111/cobi.13292

[62] Phelps M.P., Seeb L.W., Seeb J.E. Transforming ecology and conservation biology through genome editing // Conserv Biol. 2020. Vol. 34, No. 1. P. 54–65. DOI: 10.1111/cobi.13292

[63] Juergens H, Varela JA, Gorter de Vries AR, et al. Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid. FEMS Yeast Res. 2018;18(3): foy012. DOI: 10.1093/femsyr/foy012

[64] Juergens H., Varela J.A., Gorter de Vries A.R., et al. Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid // FEMS Yeast Res. 2018. Vol. 18, No. 3. ID foy012. DOI: 10.1093/femsyr/foy012

[65] Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983;166(4):557–580. DOI: 10.1016/S0022-2836(83)80284-8

[66] Hanahan D. Studies on transformation of Escherichia coli with plasmids // J Mol Biol. 1983. Vol. 166, No. 4. P. 557–580. DOI: 10.1016/S0022-2836(83)80284-8

[67] Meilhoc E, Teissie J. Electrotransformation of Saccharomyces cerevisiae. Methods Mol Biol. 2020;2050:187–193. DOI: 10.1007/978-1-4939-9740-4_21

[68] Meilhoc E., Teissie J. Electrotransformation of Saccharomyces cerevisiae // Methods Mol Biol. 2020. Vol. 2050. P. 187–193. DOI: 10.1007/978-1-4939-9740-4_21

[69] Filonov GS, Jaffrey SR. RNA Imaging with dimeric broccoli in live bacterial and mammalian cells. Curr Protoc Chem Biol. 2016;8(1): 1–28. DOI: 10.1002/9780470559277.ch150174

[70] Filonov G.S., Jaffrey S.R. RNA Imaging with dimeric Broccoli in live bacterial and mammalian cells // Curr Protoc Chem Biol. 2016. Vol. 8, No. 1. P. 1–28. DOI: 10.1002/9780470559277.ch150174

[71] Nelissen FH, Leunissen EH, van de Laar L, et al. Fast production of homogeneous recombinant RNA — towards large-scale production of RNA. Nucleic Acids Res. 2012;40(13):e102. DOI: 10.1093/nar/gks292

[72] Nelissen F.H., Leunissen E.H., van de Laar L., et al. Fast production of homogeneous recombinant RNA — towards large-scale production of RNA // Nucleic Acids Res. 2012. Vol. 40, No. 13. P. e102. DOI: 10.1093/nar/gks292

Funding

РФФИRussian Foundation for Fundamental Investigations(20-34-90139/20)

RIGHTS & PERMISSIONS

Shanaa O.A., Rumyantsev A.M., Sambuk E.V., Padkina M.V.