Home LiteratureArticle Details
PMID: 22446835 Published · epublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Rationally designed families of orthogonal RNA regulators of translation.

Nature chemical biology ·Vol. 8 ·No. 5 ·2012-03-25 ·Pages 447-54

Mutalik VK, Qi L, Guimaraes JC, Lucks JB, Arkin AP

Abstract

Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.

MeSH Terms
Base Sequence Escherichia coli/genetics Molecular Sequence Data Mutagenesis, Insertional Protein Biosynthesis RNA/metabolism RNA, Antisense/genetics Synthetic Biology/methods
Chemicals
RNA, Antisense RNA
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Mutalik Vivek K
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Qi Lei
Guimaraes Joao C
Lucks Julius B
Arkin Adam P
References (49)
49 references, click to expand
  1. The second wave of synthetic biology: from modules to systems.
    Nat Rev Mol Cell Biol. 2009 Jun;10(6):410-22 PMID: 19461664
  2. cis-antisense RNA, another level of gene regulation in bacteria.
    Microbiol Mol Biol Rev. 2011 Jun;75(2):286-300 PMID: 21646430
  3. Engineering and characterization of a superfolder green fluorescent protein.
    Nat Biotechnol. 2006 Jan;24(1):79-88 PMID: 16369541
  4. Library of synthetic 5' secondary structures to manipulate mRNA stability in Escherichia coli.
    Biotechnol Prog. 1999 Jan-Feb;15(1):58-64 PMID: 9933514
  5. Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria.
    Nucleic Acids Res. 2011 Apr;39(8):e50 PMID: 21296758
  6. Toward scalable parts families for predictable design of biological circuits.
    Curr Opin Microbiol. 2008 Dec;11(6):567-73 PMID: 18983935
  7. microRNA target predictions in animals.
    Nat Genet. 2006 Jun;38 Suppl:S8-13 PMID: 16736023
  8. Synthesis of orthogonal transcription-translation networks.
    Proc Natl Acad Sci U S A. 2009 May 26;106(21):8477-82 PMID: 19443689
  9. The IS10 transposase mRNA is destabilized during antisense RNA control.
    EMBO J. 1990 Apr;9(4):1259-66 PMID: 1691096
  10. Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli.
    Nucleic Acids Res. 2009 Aug;37(15):e103 PMID: 19515932
  11. Antisense RNAs in bacteria and their genetic elements.
    Adv Genet. 2002;46:361-98 PMID: 11931231
  12. A synthetic library of RNA control modules for predictable tuning of gene expression in yeast.
    Mol Syst Biol. 2011 Mar 1;7:471 PMID: 21364573
  13. Develop reusable and combinable designs for transcriptional logic gates.
    Mol Syst Biol. 2010 Jul 13;6:388 PMID: 20631682
  14. Models for pairing of IS10 encoded antisense RNAs in vivo.
    J Theor Biol. 1997 Jun 21;186(4):431-9 PMID: 9278719
  15. IS10 antisense control in vivo is affected by mutations throughout the region of complementarity between the interacting RNAs.
    J Mol Biol. 1995 Mar 10;246(5):585-94 PMID: 7877178
  16. Automated design of synthetic ribosome binding sites to control protein expression.
    Nat Biotechnol. 2009 Oct;27(10):946-50 PMID: 19801975
  17. A network of orthogonal ribosome x mRNA pairs.
    Nat Chem Biol. 2005 Aug;1(3):159-66 PMID: 16408021
  18. Engineered riboregulators enable post-transcriptional control of gene expression.
    Nat Biotechnol. 2004 Jul;22(7):841-7 PMID: 15208640
  19. Synthetic biology moving into the clinic.
    Science. 2011 Sep 2;333(6047):1248-52 PMID: 21885773
  20. Higher-order cellular information processing with synthetic RNA devices.
    Science. 2008 Oct 17;322(5900):456-60 PMID: 18927397
  21. Evidence for an autonomous 5' target recognition domain in an Hfq-associated small RNA.
    Proc Natl Acad Sci U S A. 2010 Nov 23;107(47):20435-40 PMID: 21059903
  22. Versatile RNA-sensing transcriptional regulators for engineering genetic networks.
    Proc Natl Acad Sci U S A. 2011 May 24;108(21):8617-22 PMID: 21555549
  23. Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure.
    J Mol Biol. 1999 Dec 17;294(5):1115-25 PMID: 10600370
  24. Organization of intracellular reactions with rationally designed RNA assemblies.
    Science. 2011 Jul 22;333(6041):470-4 PMID: 21700839
  25. Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins.
    Science. 2010 Nov 26;330(6008):1251-5 PMID: 21109673
  26. Multi-input RNAi-based logic circuit for identification of specific cancer cells.
    Science. 2011 Sep 2;333(6047):1307-11 PMID: 21885784
  27. Novel sequence-based method for identifying transcription factor binding sites in prokaryotic genomes.
    Bioinformatics. 2010 Nov 1;26(21):2672-7 PMID: 20807838
  28. Synthetic translational regulation by an L7Ae-kink-turn RNP switch.
    Nat Chem Biol. 2010 Jan;6(1):71-8 PMID: 20016495
  29. Computational design of orthogonal ribosomes.
    Nucleic Acids Res. 2008 Jul;36(12):4038-46 PMID: 18522973
  30. Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA.
    J Mol Biol. 1989 Dec 5;210(3):561-72 PMID: 2482367
  31. Specificity of microRNA target selection in translational repression.
    Genes Dev. 2004 Mar 1;18(5):504-11 PMID: 15014042
  32. BglBrick vectors and datasheets: A synthetic biology platform for gene expression.
    J Biol Eng. 2011 Sep 20;5:12 PMID: 21933410
  33. Reengineering orthogonally selective riboswitches.
    Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2830-5 PMID: 20133756
  34. Tuning genetic control through promoter engineering.
    Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12678-83 PMID: 16123130
  35. Quantitative sequence-activity models (QSAM)--tools for sequence design.
    Nucleic Acids Res. 1993 Feb 11;21(3):733-9 PMID: 8441682
  36. Regulation of transcription by unnatural amino acids.
    Nat Biotechnol. 2011 Feb;29(2):164-8 PMID: 21240267
  37. Bacterial antisense RNAs: how many are there, and what are they doing?
    Annu Rev Genet. 2010;44:167-88 PMID: 20707673
  38. Model-driven engineering of RNA devices to quantitatively program gene expression.
    Science. 2011 Dec 23;334(6063):1716-9 PMID: 22194579
  39. The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site.
    EMBO J. 1990 Apr;9(4):1267-74 PMID: 1691097
  40. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures.
    EMBO J. 1986 Nov;5(11):2987-94 PMID: 3539589
  41. Synthetic biology for synthetic chemistry.
    ACS Chem Biol. 2008 Jan 18;3(1):64-76 PMID: 18205292
  42. Multichromatic control of gene expression in Escherichia coli.
    J Mol Biol. 2011 Jan 14;405(2):315-24 PMID: 21035461
  43. RNA-based computation in live cells.
    Curr Opin Biotechnol. 2009 Aug;20(4):471-8 PMID: 19720518
  44. Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.
    Biotechnol Bioeng. 2002 May 20;78(4):412-24 PMID: 11948448
  45. Foundations for engineering biology.
    Nature. 2005 Nov 24;438(7067):449-53 PMID: 16306983
  46. UNAFold: software for nucleic acid folding and hybridization.
    Methods Mol Biol. 2008;453:3-31 PMID: 18712296
  47. A two unit antisense RNA cassette test system for silencing of target genes.
    Nucleic Acids Res. 1997 Aug 15;25(16):3218-27 PMID: 9241234
  48. Desperately seeking microRNA targets.
    Nat Struct Mol Biol. 2010 Oct;17(10):1169-74 PMID: 20924405
  49. Base pairing small RNAs and their roles in global regulatory networks.
    FEMS Microbiol Rev. 2010 Sep;34(5):866-82 PMID: 20662934
Article Info
Journal
Nature chemical biology
Abbr.
Nat Chem Biol
ISSN
1552-4469
DOI
10.1038/nchembio.919
Published
2012-03-25
Epub
2012-00-25
Pages
447-54
Language
English
Region
United States
NLM ID
101231976
Subset
IM
Corrections
CommentIn
PMID: 22510661
External Links
PubMed source DOI: 10.1038/nchembio.919 View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2