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Synthetic Biology of Small RNAs and Riboswitches

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  • Authors: Jordan K. Villa*1, Yichi Su*2, Lydia M. Contreras3,4, Ming C. Hammond5,6
  • Editors: Gisela Storz7, Kai Papenfort8
    Affiliations: 1: Institute of Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; 2: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720; 3: Institute of Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; 4: Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712; 5: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720; 6: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720; 7: Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD; 8: Department of Biology I, Microbiology, LMU Munich, Martinsried, Germany
  • Source: microbiolspec June 2018 vol. 6 no. 3 doi:10.1128/microbiolspec.RWR-0007-2017
  • Received 01 November 2017 Accepted 29 January 2018 Published 01 June 2018
  • Ming C. Hammond, [email protected]
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  • Abstract:

    In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.

  • Citation: Villa* J, Su* Y, Contreras L, Hammond M. 2018. Synthetic Biology of Small RNAs and Riboswitches. Microbiol Spectrum 6(3):RWR-0007-2017. doi:10.1128/microbiolspec.RWR-0007-2017.


1. Hallberg ZF, Su Y, Kitto RZ, Hammond MC. 2017. Engineering and in vivo applications of riboswitches. Annu Rev Biochem 86:515–539. [PubMed]
2. Vazquez-Anderson J, Contreras LM. 2013. Regulatory RNAs: charming gene management styles for synthetic biology applications. RNA Biol 10:1778–1797. [PubMed]
3. Cho SH, Haning K, Contreras LM. 2015. Strain engineering via regulatory noncoding RNAs: not a one-blueprint-fits-all. Curr Opin Chem Eng 10:25–34. [PubMed]
4. Saberi F, Kamali M, Najafi A, Yazdanparast A, Moghaddam MM. 2016. Natural antisense RNAs as mRNA regulatory elements in bacteria: a review on function and applications. Cell Mol Biol Lett 21:6. doi:10.1186/s11658-016-0007-z.
5. Etzel M, Mörl M. 2017. Synthetic riboswitches: from plug and pray toward plug and play. Biochemistry 56:1181–1198. [PubMed]
6. Kushwaha M, Rostain W, Prakash S, Duncan JN, Jaramillo A. 2016. Using RNA as molecular code for programming cellular function. ACS Synth Biol 5:795–809. [PubMed]
7. Wassarman KM, Zhang A, Storz G. 1999. Small RNAs in Escherichia coli. Trends Microbiol 7:37–45. [PubMed]
8. Papenfort K, Vanderpool CK. 2015. Target activation by regulatory RNAs in bacteria. FEMS Microbiol Rev 39:362–378. [PubMed]
9. Wagner EG, Romby P. 2015. Small RNAs in bacteria and archaea: who they are, what they do, and how they do it. Adv Genet 90:133–208. [PubMed]
10. Ellington AD, Szostak JW. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822. [PubMed]
11. Tuerk C, Gold L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510. [PubMed]
12. Soukup GA, Breaker RR. 1999. Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96:3584–3589. [PubMed]
13. Araki M, Okuno Y, Hara Y, Sugiura Y. 1998. Allosteric regulation of a ribozyme activity through ligand-induced conformational change. Nucleic Acids Res 26:3379–3384. [PubMed]
14. Gelfand MS, Mironov AA, Jomantas J, Kozlov YI, Perumov DA. 1999. A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet 15:439–442.
15. Nou X, Kadner RJ. 1998. Coupled changes in translation and transcription during cobalamin-dependent regulation of btuB expression in Escherichia coli. J Bacteriol 180:6719–6728. [PubMed]
16. Miranda-Ríos J, Navarro M, Soberón M. 2001. A conserved RNA structure ( thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria. Proc Natl Acad Sci U S A 98:9736–9741. [PubMed]
17. Mironov AS, Gusarov I, Rafikov R, Lopez LE, Shatalin K, Kreneva RA, Perumov DA, Nudler E. 2002. Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111:747–756. [PubMed]
18. Nahvi A, Sudarsan N, Ebert MS, Zou X, Brown KL, Breaker RR. 2002. Genetic control by a metabolite binding mRNA. Chem Biol 9:1043–1049. [PubMed]
19. Winkler WC, Cohen-Chalamish S, Breaker RR. 2002. An mRNA structure that controls gene expression by binding FMN. Proc Natl Acad Sci U S A 99:15908–15913. [PubMed]
20. Winkler W, Nahvi A, Breaker RR. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419:952–956. [PubMed]
21. Bandyra KJ, Said N, Pfeiffer V, Górna MW, Vogel J, Luisi BF. 2012. The seed region of a small RNA drives the controlled destruction of the target mRNA by the endoribonuclease RNase E. Mol Cell 47:943–953. [PubMed]
22. Hollands K, Proshkin S, Sklyarova S, Epshtein V, Mironov A, Nudler E, Groisman EA. 2012. Riboswitch control of Rho-dependent transcription termination. Proc Natl Acad Sci U S A 109:5376–5381. [PubMed]
23. Serganov A, Nudler E. 2013. A decade of riboswitches. Cell 152:17–24. [PubMed]
24. Cavanagh AT, Wassarman KM. 2014. 6S RNA, a global regulator of transcription in Escherichia coli, Bacillus subtilis, and beyond. Annu Rev Microbiol 68:45–60. [PubMed]
25. Sedlyarova N, Shamovsky I, Bharati BK, Epshtein V, Chen J, Gottesman S, Schroeder R, Nudler E. 2016. sRNA-mediated control of transcription termination in E. coli. Cell 167:111–121.e13. [PubMed]
26. Sedlyarova N, Rescheneder P, Magán A, Popitsch N, Rziha N, Bilusic I, Epshtein V, Zimmermann B, Lybecker M, Sedlyarov V, Schroeder R, Nudler E. 2017. Natural RNA polymerase aptamers regulate transcription in E. coli. Mol Cell 67:30–43.e6. doi:10.1016/j.molcel.2017.05.025. [PubMed]
27. Lemay JF, Desnoyers G, Blouin S, Heppell B, Bastet L, St-Pierre P, Massé E, Lafontaine DA. 2011. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms. PLoS Genet 7:e1001278. doi:10.1371/journal.pgen.1001278. [PubMed]
28. Sudarsan N, Lee ER, Weinberg Z, Moy RH, Kim JN, Link KH, Breaker RR. 2008. Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321:411–413. [PubMed]
29. Lee ER, Baker JL, Weinberg Z, Sudarsan N, Breaker RR. 2010. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329:845–848. [PubMed]
30. Gorski SA, Vogel J, Doudna JA. 2017. RNA-based recognition and targeting: sowing the seeds of specificity. Nat Rev Mol Cell Biol 18:215–228. [PubMed]
31. Kawamoto H, Koide Y, Morita T, Aiba H. 2006. Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq. Mol Microbiol 61:1013–1022. [PubMed]
32. Mizuno T, Chou MY, Inouye M. 1984. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A 81:1966–1970. [PubMed]
33. Vazquez-Anderson J, Mihailovic MK, Baldridge KC, Reyes KG, Haning K, Cho SH, Amador P, Powell WB, Contreras LM. 2017. Optimization of a novel biophysical model using large scale in vivo antisense hybridization data displays improved prediction capabilities of structurally accessible RNA regions. Nucleic Acids Res 45:5523–5538. [PubMed]
34. Wassarman KM, Repoila F, Rosenow C, Storz G, Gottesman S. 2001. Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev 15:1637–1651. [PubMed]
35. Carter RJ, Dubchak I, Holbrook SR. 2001. A computational approach to identify genes for functional RNAs in genomic sequences. Nucleic Acids Res 29:3928–3938. [PubMed]
36. Livny J, Waldor MK. 2007. Identification of small RNAs in diverse bacterial species. Curr Opin Microbiol 10:96–101. [PubMed]
37. Babski J, Maier LK, Heyer R, Jaschinski K, Prasse D, Jäger D, Randau L, Schmitz RA, Marchfelder A, Soppa J. 2014. Small regulatory RNAs in Archaea. RNA Biol 11:484–493. [PubMed]
38. Sharma CM, Vogel J. 2009. Experimental approaches for the discovery and characterization of regulatory small RNA. Curr Opin Microbiol 12:536–546. [PubMed]
39. Tsai CH, Liao R, Chou B, Palumbo M, Contreras LM. 2015. Genome-wide analyses in bacteria show small-RNA enrichment for long and conserved intergenic regions. J Bacteriol 197:40–50. [PubMed]
40. Gelderman G, Contreras LM. 2013. Discovery of posttranscriptional regulatory RNAs using next generation sequencing technologies. Methods Mol Biol 985:269–295. [PubMed]
41. DeJesus MA, Gerrick ER, Xu W, Park SW, Long JE, Boutte CC, Rubin EJ, Schnappinger D, Ehrt S, Fortune SM, Sassetti CM, Ioerger TR. 2017. Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis. mBio 8:e02133-16. doi:10.1128/mBio.02133-16. [PubMed]
42. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K, Hackermüller J, Reinhardt R, Stadler PF, Vogel J. 2010. The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464:250–255. [PubMed]
43. Fakhry CT, Kulkarni P, Chen P, Kulkarni R, Zarringhalam K. 2017. Prediction of bacterial small RNAs in the RsmA (CsrA) and ToxT pathways: a machine learning approach. BMC Genomics 18:645. doi:10.1186/s12864-017-4057-z. [PubMed]
44. Barrick JE, Corbino KA, Winkler WC, Nahvi A, Mandal M, Collins J, Lee M, Roth A, Sudarsan N, Jona I, Wickiser JK, Breaker RR. 2004. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc Natl Acad Sci U S A 101:6421–6426. [PubMed]
45. Weinberg Z, Barrick JE, Yao Z, Roth A, Kim JN, Gore J, Wang JX, Lee ER, Block KF, Sudarsan N, Neph S, Tompa M, Ruzzo WL, Breaker RR. 2007. Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline. Nucleic Acids Res 35:4809–4819. [PubMed]
46. Yao Z, Barrick J, Weinberg Z, Neph S, Breaker R, Tompa M, Ruzzo WL. 2007. A computational pipeline for high-throughput discovery of cis-regulatory noncoding RNA in prokaryotes. PLoS Comput Biol 3:e126. doi:10.1371/journal.pcbi.0030126. [PubMed]
47. Weinberg Z, Wang JX, Bogue J, Yang J, Corbino K, Moy RH, Breaker RR. 2010. Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaea, and their metagenomes. Genome Biol 11:R31. doi:10.1186/gb-2010-11-3-r31. [PubMed]
48. McCown PJ, Corbino KA, Stav S, Sherlock ME, Breaker RR. 2017. Riboswitch diversity and distribution. RNA 23:995–1011. [PubMed]
49. Rosinski-Chupin I, Sauvage E, Sismeiro O, Villain A, Da Cunha V, Caliot ME, Dillies MA, Trieu-Cuot P, Bouloc P, Lartigue MF, Glaser P. 2015. Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae. BMC Genomics 16:419. doi:10.1186/s12864-015-1583-4. [PubMed]
50. Rosinski-Chupin I, Soutourina O, Martin-Verstraete I. 2014. Riboswitch discovery by combining RNA-seq and genome-wide identification of transcriptional start sites. Methods Enzymol 549:3–27. [PubMed]
51. Sledjeski D, Gottesman S. 1995. A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli. Proc Natl Acad Sci U S A 92:2003–2007. [PubMed]
52. Lalaouna D, Massé E. 2016. The spectrum of activity of the small RNA DsrA: not so narrow after all. Curr Genet 62:261–264. [PubMed]
53. Melamed S, Peer A, Faigenbaum-Romm R, Gatt YE, Reiss N, Bar A, Altuvia Y, Argaman L, Margalit H. 2016. Global mapping of small RNA-target interactions in bacteria. Mol Cell 63:884–897. [PubMed]
54. Liu T, Zhang K, Xu S, Wang Z, Fu H, Tian B, Zheng X, Li W. 2017. Detecting RNA-RNA interactions in E. coli using a modified CLASH method. BMC Genomics 18:343. doi:10.1186/s12864-017-3725-3. [PubMed]
55. Wang J, Rennie W, Liu C, Carmack CS, Prévost K, Caron MP, Massé E, Ding Y, Wade JT. 2015. Identification of bacterial sRNA regulatory targets using ribosome profiling. Nucleic Acids Res 43:10308–10320.
56. Bourqui R, Dutour I, Dubois J, Benchimol W, Thébault P. 2017. rNAV 2.0: a visualization tool for bacterial sRNA-mediated regulatory networks mining. BMC Bioinformatics 18:188. doi:10.1186/s12859-017-1598-8. [PubMed]
57. Wang J, Liu T, Zhao B, Lu Q, Wang Z, Cao Y, Li W. 2016. sRNATarBase 3.0: an updated database for sRNA-target interactions in bacteria. Nucleic Acids Res 44(D1) :D248–D253. [PubMed]
58. Ivain L, Bordeau V, Eyraud A, Hallier M, Dreano S, Tattevin P, Felden B, Chabelskaya S. 2017. An in vivo reporter assay for sRNA-directed gene control in Gram-positive bacteria: identifying a novel sRNA target in Staphylococcus aureus. Nucleic Acids Res 45:4994–5007. [PubMed]
59. Jagodnik J, Brosse A, Le Lam TN, Chiaruttini C, Guillier M. 2017. Mechanistic study of base-pairing small regulatory RNAs in bacteria. Methods 117:67–76. [PubMed]
60. Nelson JW, Sudarsan N, Furukawa K, Weinberg Z, Wang JX, Breaker RR. 2013. Riboswitches in eubacteria sense the second messenger c-di-AMP. Nat Chem Biol 9:834–839. [PubMed]
61. Nelson JW, Sudarsan N, Phillips GE, Stav S, Lünse CE, McCown PJ, Breaker RR. 2015. Control of bacterial exoelectrogenesis by c-AMP-GMP. Proc Natl Acad Sci U S A 112:5389–5394. [PubMed]
62. Kellenberger CA, Wilson SC, Hickey SF, Gonzalez TL, Su Y, Hallberg ZF, Brewer TF, Iavarone AT, Carlson HK, Hsieh YF, Hammond MC. 2015. GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP. Proc Natl Acad Sci U S A 112:5383–5388. [PubMed]
63. Hallberg ZF, Wang XC, Wright TA, Nan B, Ad O, Yeo J, Hammond MC. 2016. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP). Proc Natl Acad Sci U S A 113:1790–1795. [PubMed]
64. Meyer MM, Roth A, Chervin SM, Garcia GA, Breaker RR. 2008. Confirmation of a second natural preQ 1 aptamer class in Streptococcaceae bacteria. RNA 14:685–695. [PubMed]
65. Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335:233–235. [PubMed]
66. Nelson JW, Atilho RM, Sherlock ME, Stockbridge RB, Breaker RR. 2017. Metabolism of free guanidine in bacteria is regulated by a widespread riboswitch class. Mol Cell 65:220–230. [PubMed]
67. Sherlock ME, Breaker RR. 2017. Biochemical validation of a third guanidine riboswitch class in bacteria. Biochemistry 56:359–363. [PubMed]
68. Mandal M, Breaker RR. 2004. Adenine riboswitches and gene activation by disruption of a transcription terminator. Nat Struct Mol Biol 11:29–35. [PubMed]
69. Kim JN, Roth A, Breaker RR. 2007. Guanine riboswitch variants from Mesoplasma florum selectively recognize 2′-deoxyguanosine. Proc Natl Acad Sci U S A 104:16092–16097. [PubMed]
70. Weinberg Z, Nelson JW, Lünse CE, Sherlock ME, Breaker RR. 2017. Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity. Proc Natl Acad Sci U S A 114:E2077–E2085. [PubMed]
71. Kumari P, Sampath K. 2015. cncRNAs: bi-functional RNAs with protein coding and non-coding functions. Semin Cell Dev Biol 47–48:40–51. [PubMed]
72. Bronsard J, Pascreau G, Sassi M, Mauro T, Augagneur Y, Felden B. 2017. sRNA and cis-antisense sRNA identification in Staphylococcus aureus highlights an unusual sRNA gene cluster with one encoding a secreted peptide. Sci Rep 7:4565. doi:10.1038/s41598-017-04786-3. [PubMed]
73. Wadler CS, Vanderpool CK. 2007. A dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide. Proc Natl Acad Sci U S A 104:20454–20459. [PubMed]
74. Lloyd CR, Park S, Fei J, Vanderpool CK. 2017. The small protein SgrT controls transport activity of the glucose-specific phosphotransferase system. J Bacteriol 199:1–14. [PubMed]
75. Lago M, Monteil V, Douche T, Guglielmini J, Criscuolo A, Maufrais C, Matondo M, Norel F. 2017. Proteome remodelling by the stress sigma factor RpoS/σ S in Salmonella: identification of small proteins and evidence for post-transcriptional regulation. Sci Rep 7:2127. doi:10.1038/s41598-017-02362-3. [PubMed]
76. Savinov A, Perez CF, Block SM. 2014. Single-molecule studies of riboswitch folding. Biochim Biophys Acta 1839:1030–1045. [PubMed]
77. Haller A, Rieder U, Aigner M, Blanchard SC, Micura R. 2011. Conformational capture of the SAM-II riboswitch. Nat Chem Biol 7:393–400. [PubMed]
78. Heppell B, Blouin S, Dussault AM, Mulhbacher J, Ennifar E, Penedo JC, Lafontaine DA. 2011. Molecular insights into the ligand-controlled organization of the SAM-I riboswitch. Nat Chem Biol 7:384–392. [PubMed]
79. Zhao B, Guffy SL, Williams B, Zhang Q. 2017. An excited state underlies gene regulation of a transcriptional riboswitch. Nat Chem Biol 13:968–974. [PubMed]
80. Hammond MC. 2011. RNA folding: a tale of two riboswitches. Nat Chem Biol 7:342–343. [PubMed]
81. Manz C, Kobitski AY, Samanta A, Keller BG, Jäschke A, Nienhaus GU. 2017. Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch. Nat Chem Biol 13:1172–1178. [PubMed]
82. Watters KE, Strobel EJ, Yu AM, Lis JT, Lucks JB. 2016. Cotranscriptional folding of a riboswitch at nucleotide resolution. Nat Struct Mol Biol 23:1124–1131. [PubMed]
83. Updegrove TB, Zhang A, Storz G. 2016. Hfq: the flexible RNA matchmaker. Curr Opin Microbiol 30:133–138. [PubMed]
84. Schu DJ, Zhang A, Gottesman S, Storz G. 2015. Alternative Hfq-sRNA interaction modes dictate alternative mRNA recognition. EMBO J 34:2557–2573. [PubMed]
85. Santiago-Frangos A, Kavita K, Schu DJ, Gottesman S, Woodson SA. 2016. C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA. Proc Natl Acad Sci U S A 113:E6089–E6096. [PubMed]
86. Malabirade A, Morgado-Brajones J, Trépout S, Wien F, Marquez I, Seguin J, Marco S, Velez M, Arluison V. 2017. Membrane association of the bacterial riboregulator Hfq and functional perspectives. Sci Rep 7:10724. doi:10.1038/s41598-017-11157-5. [PubMed]
87. Bouloc P, Repoila F. 2016. Fresh layers of RNA-mediated regulation in Gram-positive bacteria. Curr Opin Microbiol 30:30–35. [PubMed]
88. Nielsen JS, Lei LK, Ebersbach T, Olsen AS, Klitgaard JK, Valentin-Hansen P, Kallipolitis BH. 2010. Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes. Nucleic Acids Res 38:907–919. [PubMed]
89. Olejniczak M, Storz G. 2017. ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers? Mol Microbiol 104:905–915. [PubMed]
90. Wickiser JK, Winkler WC, Breaker RR, Crothers DM. 2005. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49–60. [PubMed]
91. Espah Borujeni A, Mishler DM, Wang J, Huso W, Salis HM. 2016. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers. Nucleic Acids Res 44:1–13. [PubMed]
92. Adamson DN, Lim HN. 2011. Essential requirements for robust signaling in Hfq dependent small RNA networks. PLoS Comput Biol 7:e1002138. doi:10.1371/journal.pcbi.1002138. [PubMed]
93. Bossi L, Figueroa-Bossi N. 2016. Competing endogenous RNAs: a target-centric view of small RNA regulation in bacteria. Nat Rev Microbiol 14:775–784. [PubMed]
94. Gaida SM, Al-Hinai MA, Indurthi DC, Nicolaou SA, Papoutsakis ET. 2013. Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress. Nucleic Acids Res 41:8726–8737. [PubMed]
95. Mandal M, Lee M, Barrick JE, Weinberg Z, Emilsson GM, Ruzzo WL, Breaker RR. 2004. A glycine-dependent riboswitch that uses cooperative binding to control gene expression. Science 306:275–279. [PubMed]
96. Welz R, Breaker RR. 2007. Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis. RNA 13:573–582. [PubMed]
97. Zhou H, Zheng C, Su J, Chen B, Fu Y, Xie Y, Tang Q, Chou SH, He J. 2016. Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter. Sci Rep 6:20871. doi:10.1038/srep20871. [PubMed]
98. Sudarsan N, Hammond MC, Block KF, Welz R, Barrick JE, Roth A, Breaker RR. 2006. Tandem riboswitch architectures exhibit complex gene control functions. Science 314:300–304. [PubMed]
99. Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR. 2004. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428:281–286. [PubMed]
100. Klein DJ, Ferré-D’Amaré AR. 2006. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313:1752–1756. [PubMed]
101. Cochrane JC, Lipchock SV, Smith KD, Strobel SA. 2009. Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme. Biochemistry 48:3239–3246. [PubMed]
102. Cheah MT, Wachter A, Sudarsan N, Breaker RR. 2007. Control of alternative RNA splicing and gene expression by eukaryotic riboswitches. Nature 447:497–500. [PubMed]
103. DebRoy S, Gebbie M, Ramesh A, Goodson JR, Cruz MR, van Hoof A, Winkler WC, Garsin DA. 2014. Riboswitches. A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator. Science 345:937–940. [PubMed]
104. Loh E, Dussurget O, Gripenland J, Vaitkevicius K, Tiensuu T, Mandin P, Repoila F, Buchrieser C, Cossart P, Johansson J. 2009. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell 139:770–779. [PubMed]
105. Lahiry A, Stimple SD, Wood DW, Lease RA. 2017. Retargeting a dual-acting sRNA for multiple mRNA transcript regulation. ACS Synth Biol 6:648–658. [PubMed]
106. Hoynes-O’Connor A, Moon TS. 2016. Development of design rules for reliable antisense RNA behavior in E. coli. ACS Synth Biol 5:1441–1454. [PubMed]
107. Papenfort K, Bouvier M, Mika F, Sharma CM, Vogel J. 2010. Evidence for an autonomous 5′ target recognition domain in an Hfq-associated small RNA. Proc Natl Acad Sci U S A 107:20435–20440. [PubMed]
108. Fröhlich KS, Papenfort K, Fekete A, Vogel J. 2013. A small RNA activates CFA synthase by isoform-specific mRNA stabilization. EMBO J 32:2963–2979. [PubMed]
109. Noro E, Mori M, Makino G, Takai Y, Ohnuma S, Sato A, Tomita M, Nakahigashi K, Kanai A. 2017. Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth. RNA Biol 14:206–218. [PubMed]
110. Sharma V, Yamamura A, Yokobayashi Y. 2012. Engineering artificial small RNAs for conditional gene silencing in Escherichia coli. ACS Synth Biol 1:6–13. [PubMed]
111. Wasmuth EV, Lima CD. 2017. The Rrp6 C-terminal domain binds RNA and activates the nuclear RNA exosome. Nucleic Acids Res 45:846–860. [PubMed]
112. Lee YJ, Moon TS. 2018. Design rules of synthetic non-coding RNAs in bacteria. Methods S1046-2023(17)30338-9. doi:10.1016/j.ymeth.2018.01.001. [PubMed]
113. Man S, Cheng R, Miao C, Gong Q, Gu Y, Lu X, Han F, Yu W. 2011. Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria. Nucleic Acids Res 39:e50. doi:10.1093/nar/gkr034. [PubMed]
114. Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. 2013. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol 31:170–174. [PubMed]
115. Leistra AN, Amador P, Buvanendiran A, Moon-Walker A, Contreras LM. 2017. Rational modular RNA engineering based on in vivo profiling of structural accessibility. ACS Synth Biol 6:2228–2240. [PubMed]
116. Jenison RD, Gill SC, Pardi A, Polisky B. 1994. High-resolution molecular discrimination by RNA. Science 263:1425–1429. [PubMed]
117. Desai SK, Gallivan JP. 2004. Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. J Am Chem Soc 126:13247–13254. [PubMed]
118. Suess B, Fink B, Berens C, Stentz R, Hillen W. 2004. A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res 32:1610–1614. [PubMed]
119. Goler JA, Carothers JM, Keasling JD. 2014. Dual-selection for evolution of in vivo functional aptazymes as riboswitch parts. Methods Mol Biol 1111:221–235. [PubMed]
120. Dixon N, Duncan JN, Geerlings T, Dunstan MS, McCarthy JE, Leys D, Micklefield J. 2010. Reengineering orthogonally selective riboswitches. Proc Natl Acad Sci U S A 107:2830–2835. [PubMed]
121. Nomura Y, Yokobayashi Y. 2007. Reengineering a natural riboswitch by dual genetic selection. J Am Chem Soc 129:13814–13815. [PubMed]
122. Sparvath SL, Geary CW, Andersen ES. 2017. Computer-aided design of RNA origami structures. Methods Mol Biol 1500:51–80. [PubMed]
123. Cho C, Lee SY. 2017. Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs. Biotechnol Bioeng 114:374–383. [PubMed]
124. Venkataramanan KP, Jones SW, McCormick KP, Kunjeti SG, Ralston MT, Meyers BC, Papoutsakis ET. 2013. The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum. BMC Genomics 14:849. doi:10.1186/1471-2164-14-849. [PubMed]
125. Cho SH, Lei R, Henninger TD, Contreras LM. 2014. Discovery of ethanol-responsive small RNAs in Zymomonas mobilis. Appl Environ Microbiol 80:4189–4198. [PubMed]
126. Jones AJ, Venkataramanan KP, Papoutsakis T. 2016. Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum. FEMS Microbiol Lett 363:1–6. [PubMed]
127. Pei G, Sun T, Chen S, Chen L, Zhang W. 2017. Systematic and functional identification of small non-coding RNAs associated with exogenous biofuel stress in cyanobacterium Synechocystis sp. PCC 6803. Biotechnol Biofuels 10:57. doi:10.1186/s13068-017-0743-y. [PubMed]
128. Liu M, Zhu ZT, Tao XY, Wang FQ, Wei DZ. 2016. RNA-seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates. Microb Cell Fact 15:64. doi:10.1186/s12934-016-0462-2. [PubMed]
129. Hertel R, Meyerjürgens S, Voigt B, Liesegang H, Volland S. 2017. Small RNA mediated repression of subtilisin production in Bacillus licheniformis. Sci Rep 7:5699. doi:10.1038/s41598-017-05628-y. [PubMed]
130. Zess EK, Begemann MB, Pfleger BF. 2016. Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. Biotechnol Bioeng 113:424–432. [PubMed]
131. Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis T. 2016. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc Natl Acad Sci U S A 113:E3431–E3440. [PubMed]
132. Zhou LB, Zeng AP. 2015. Exploring lysine riboswitch for metabolic flux control and improvement of l-lysine synthesis in Corynebacterium glutamicum. ACS Synth Biol 4:729–734. [PubMed]
133. Zhou LB, Zeng AP. 2015. Engineering a lysine-ON riboswitch for metabolic control of lysine production in Corynebacterium glutamicum. ACS Synth Biol 4:1335–1340. [PubMed]
134. Wang J, Gao D, Yu X, Li W, Qi Q. 2015. Evolution of a chimeric aspartate kinase for l-lysine production using a synthetic RNA device. Appl Microbiol Biotechnol 99:8527–8536. [PubMed]
135. Yang J, Seo SW, Jang S, Shin SI, Lim CH, Roh TY, Jung GY. 2013. Synthetic RNA devices to expedite the evolution of metabolite-producing microbes. Nat Commun 4:1413. doi:10.1038/ncomms2404. [PubMed]
136. Meyer A, Pellaux R, Potot S, Becker K, Hohmann HP, Panke S, Held M. 2015. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nat Chem 7:673–678. [PubMed]
137. Vincent HA, Robinson CJ, Wu MC, Dixon N, Micklefield J. 2014. Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening. Methods Mol Biol 1111:107–129. [PubMed]
138. Robinson CJ, Vincent HA, Wu MC, Lowe PT, Dunstan MS, Leys D, Micklefield J. 2014. Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species. J Am Chem Soc 136:10615–10624. [PubMed]
139. Wu MC, Lowe PT, Robinson CJ, Vincent HA, Dixon N, Leigh J, Micklefield J. 2015. Rational re-engineering of a transcriptional silencing PreQ 1 riboswitch. J Am Chem Soc 137:9015–9021. [PubMed]
140. Topp S, Reynoso CM, Seeliger JC, Goldlust IS, Desai SK, Murat D, Shen A, Puri AW, Komeili A, Bertozzi CR, Scott JR, Gallivan JP. 2010. Synthetic riboswitches that induce gene expression in diverse bacterial species. Appl Environ Microbiol 76:7881–7884. [PubMed]
141. Bugrysheva JV, Froehlich BJ, Freiberg JA, Scott JR. 2011. The histone-like protein Hlp is essential for growth of Streptococcus pyogenes: comparison of genetic approaches to study essential genes. Appl Environ Microbiol 77:4422–4428. [PubMed]
142. Reynoso CM, Miller MA, Bina JE, Gallivan JP, Weiss DS. 2012. Riboswitches for intracellular study of genes involved in Francisella pathogenesis. mBio 3:e00253-12. doi:10.1128/mBio.00253-12. [PubMed]
143. Seeliger JC, Topp S, Sogi KM, Previti ML, Gallivan JP, Bertozzi CR. 2012. A riboswitch-based inducible gene expression system for mycobacteria. PLoS One 7:e29266. doi:10.1371/journal.pone.0029266. [PubMed]
144. Rudolph MM, Vockenhuber MP, Suess B. 2015. Conditional control of gene expression by synthetic riboswitches in Streptomyces coelicolor. Methods Enzymol 550:283–299. [PubMed]
145. Rudolph MM, Vockenhuber MP, Suess B. 2013. Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. Microbiology 159:1416–1422. [PubMed]
146. Nakahira Y, Ogawa A, Asano H, Oyama T, Tozawa Y. 2013. Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in cyanobacterium Synechococcus elongatus PCC 7942. Plant Cell Physiol 54:1724–1735. [PubMed]
147. Dwidar M, Yokobayashi Y. 2017. Controlling Bdellovibrio bacteriovorus gene expression and predation using synthetic riboswitches. ACS Synth Biol 6:2035–2041. [PubMed]
148. Ma AT, Schmidt CM, Golden JW. 2014. Regulation of gene expression in diverse cyanobacterial species by using theophylline-responsive riboswitches. Appl Environ Microbiol 80:6704–6713. [PubMed]
149. Gelderman G, Sivakumar A, Lipp S, Contreras L. 2015. Adaptation of tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria. Biotechnol Bioeng 112:365–375. [PubMed]
150. Alam KK, Tawiah KD, Lichte MF, Porciani D, Burke DH. 2017. A fluorescent split aptamer for visualizing RNA-RNA assembly in vivo. ACS Synth Biol 6:1710–1721. [PubMed]
151. Sowa SW, Vazquez-Anderson J, Clark CA, De La Peña R, Dunn K, Fung EK, Khoury MJ, Contreras LM. 2015. Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence. Nucleic Acids Res 43:e13. doi:10.1093/nar/gku1191. [PubMed]
152. Fowler CC, Brown ED, Li Y. 2010. Using a riboswitch sensor to examine coenzyme B 12 metabolism and transport in E. coli. Chem Biol 17:756–765. [PubMed]
153. Fowler CC, Sugiman-Marangos S, Junop MS, Brown ED, Li Y. 2013. Exploring intermolecular interactions of a substrate binding protein using a riboswitch-based sensor. Chem Biol 20:1502–1512. [PubMed]
154. Gao X, Dong X, Subramanian S, Matthews PM, Cooper CA, Kearns DB, Dann CE III. 2014. Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases. Appl Environ Microbiol 80:6167–6174. [PubMed]
155. Paige JS, Wu KY, Jaffrey SR. 2011. RNA mimics of green fluorescent protein. Science 333:642–646. [PubMed]
156. Kellenberger CA, Wilson SC, Sales-Lee J, Hammond MC. 2013. RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J Am Chem Soc 135:4906–4909. [PubMed]
157. Wang XC, Wilson SC, Hammond MC. 2016. Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP. Nucleic Acids Res 44:e139. doi:10.1093/nar/gkw580. [PubMed]
158. Su Y, Hickey SF, Keyser SG, Hammond MC. 2016. In vitro and in vivo enzyme activity screening via RNA-based fluorescent biosensors for S-adenosyl- l-homocysteine (SAH). J Am Chem Soc 138:7040–7047. [PubMed]
159. Kellenberger CA, Chen C, Whiteley AT, Portnoy DA, Hammond MC. 2015. RNA-based fluorescent biosensors for live cell imaging of second messenger cyclic di-AMP. J Am Chem Soc 137:6432–6435. [PubMed]
160. Bose D, Su Y, Marcus A, Raulet DH, Hammond MC. 2016. An RNA-based fluorescent biosensor for high-throughput analysis of the cGAS-cGAMP-STING pathway. Cell Chem Biol 23:1539–1549. [PubMed]
161. Yeo J, Dippel AB, Wang XC, Hammond MC. 2018. In vivo biochemistry: single-cell dynamics of cyclic di-GMP in Escherichia coli in response to zinc overload. Biochemistry 57:108–116. [PubMed]
162. Papenfort K, Espinosa E, Casadesús J, Vogel J. 2015. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella. Proc Natl Acad Sci U S A 112:E4772–E4781. [PubMed]
163. Green AA, Kim J, Ma D, Silver PA, Collins JJ, Yin P. 2017. Complex cellular logic computation using ribocomputing devices. Nature 548:117–121. [PubMed]
164. Green AA, Silver PA, Collins JJ, Yin P. 2014. Toehold switches: de-novo-designed regulators of gene expression. Cell 159:925–939. [PubMed]
165. Chappell J, Takahashi MK, Lucks JB. 2015. Creating small transcription activating RNAs. Nat Chem Biol 11:214–220. [PubMed]
166. Wachsmuth M, Domin G, Lorenz R, Serfling R, Findeiß S, Stadler PF, Mörl M. 2015. Design criteria for synthetic riboswitches acting on transcription. RNA Biol 12:221–231. [PubMed]
167. Domin G, Findeiß S, Wachsmuth M, Will S, Stadler PF, Mörl M. 2017. Applicability of a computational design approach for synthetic riboswitches. Nucleic Acids Res 45:4108–4119. [PubMed]
168. Sharma V, Nomura Y, Yokobayashi Y. 2008. Engineering complex riboswitch regulation by dual genetic selection. J Am Chem Soc 130:16310–16315. [PubMed]
169. Jakočiūnas T, Jensen MK, Keasling JD. 2016. CRISPR/Cas9 advances engineering of microbial cell factories. Metab Eng 34:44–59. [PubMed]
170. Haeussler M, Concordet JP. 2016. Genome editing with CRISPR-Cas9: can it get any better? J Genet Genomics 43:239–250. [PubMed]
171. Dersch P, Khan MA, Mühlen S, Görke B. 2017. Roles of regulatory RNAs for antibiotic resistance in bacteria and their potential value as novel drug targets. Front Microbiol 8:803. doi:10.3389/fmicb.2017.00803. [PubMed]
172. Jakobsen TH, Warming AN, Vejborg RM, Moscoso JA, Stegger M, Lorenzen F, Rybtke M, Andersen JB, Petersen R, Andersen PS, Nielsen TE, Tolker-Nielsen T, Filloux A, Ingmer H, Givskov M. 2017. A broad range quorum sensing inhibitor working through sRNA inhibition. Sci Rep 7:9857. doi:10.1038/s41598-017-09886-8. [PubMed]
173. Howe JA, Wang H, Fischmann TO, Balibar CJ, Xiao L, Galgoci AM, Malinverni JC, Mayhood T, Villafania A, Nahvi A, Murgolo N, Barbieri CM, Mann PA, Carr D, Xia E, Zuck P, Riley D, Painter RE, Walker SS, Sherborne B, de Jesus R, Pan W, Plotkin MA, Wu J, Rindgen D, Cummings J, Garlisi CG, Zhang R, Sheth PR, Gill CJ, Tang H, Roemer T. 2015. Selective small-molecule inhibition of an RNA structural element. Nature 526:672–677. [PubMed]
174. Jasinski D, Haque F, Binzel DW, Guo P. 2017. Advancement of the emerging field of RNA nanotechnology. ACS Nano 11:1142–1164. [PubMed]
175. Stewart JM, Subramanian HK, Franco E. 2017. Self-assembly of multi-stranded RNA motifs into lattices and tubular structures. Nucleic Acids Res 45:5449–5457. [PubMed]
176. Bui MN, Brittany Johnson M, Viard M, Satterwhite E, Martins AN, Li Z, Marriott I, Afonin KA, Khisamutdinov EF. 2017. Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology. Nanomedicine (Lond) 13:1137–1146. [PubMed]
177. Oi H, Fujita D, Suzuki Y, Sugiyama H, Endo M, Matsumura S, Ikawa Y. 2017. Programmable formation of catalytic RNA triangles and squares by assembling modular RNA enzymes. J Biochem 161:451–462. [PubMed]
178. Gallagher RR, Patel JR, Interiano AL, Rovner AJ, Isaacs FJ. 2015. Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Res 43:1945–1954. [PubMed]
179. Pardee K, Green AA, Ferrante T, Cameron DE, DaleyKeyser A, Yin P, Collins JJ. 2014. Paper-based synthetic gene networks. Cell 159:940–954. [PubMed]

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In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.

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Image of FIGURE 1

Timeline of sRNA and riboswitch discovery, including relevant technological advances that aided identification and verification of regulatory RNAs. The development of high-throughput, deep-sequencing techniques in particular has led to an explosion of sRNA and riboswitch discovery. However, although identification of sRNAs and riboswitches has rapidly expanded, verification of function still lags behind.

Source: microbiolspec June 2018 vol. 6 no. 3 doi:10.1128/microbiolspec.RWR-0007-2017
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Image of FIGURE 2

General function of sRNAs (A to D) and riboswitches (a to f). sRNAs regulate gene expression in through several functions enacted by antisense interactions, including transcription attenuation/enhancement through interactions with the RNA polymerase (A), inhibition of protein or ribosome binding either indirectly (B) or directly (C), and sequestration of protein factors (such as CsrA) (D). Riboswitches regulate gene expression in through a ligand-induced conformational change in the expression platform. The resulting gene expression consequences include Rho-dependent/independent transcription termination (a, b), transcription antitermination (c), translation activation (d), translation inhibition (e), and mRNA degradation (f).

Source: microbiolspec June 2018 vol. 6 no. 3 doi:10.1128/microbiolspec.RWR-0007-2017
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Image of FIGURE 3

Examples of applications of sRNAs and riboswitches. Applications of these regulatory RNAs are rooted in their unique functional characteristics (antisense interactions for sRNA and ligand binding for riboswitches). Recent applications of these systems have begun to interweave these mechanisms to provide more complex engineering strategies.

Source: microbiolspec June 2018 vol. 6 no. 3 doi:10.1128/microbiolspec.RWR-0007-2017
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General considerations for synthetic design: comparison of general factors to be considered in synthetic applications of small regulatory RNAs and riboswitches

Source: microbiolspec June 2018 vol. 6 no. 3 doi:10.1128/microbiolspec.RWR-0007-2017

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