Computational Sequence Design with R2oDNA Designer

  • James T. MacDonaldEmail author
  • Velia Siciliano
Part of the Methods in Molecular Biology book series (MIMB, volume 1651)


Recently developed DNA assembly methods have enabled the rapid and simultaneous assembly of multiple parts to create complex synthetic gene circuits. A number of groups have proposed the use of computationally designed orthogonal spacer sequences to guide the ordered assembly of parts using overlap-directed or homologous recombination-based methods. This approach is particularly useful for assembling multiple parts with repetitive elements. Orthogonal spacer sequences (sometimes called UNSs—unique nucleotide sequences) also have a number of other potential uses including in the design of synthetic promoters regulated by novel regulatory elements.

Key words

Unique nucleotide sequences (UNSs) Spacer sequences Biologically neutral sequences Computational design DNA assembly Orthogonal sequences 



This work was funded by the Engineering and Physical Sciences Research Council, UK (EPSRC, UK), and a Junior Research Fellowship (JRF) from Imperial College London.


  1. 1.
    Siciliano V, Garzilli I, Fracassi C et al (2013) MiRNAs confer phenotypic robustness to gene networks by suppressing biological noise. Nat Commun 4:2364. doi: 10.1038/ncomms3364CrossRefPubMedGoogle Scholar
  2. 2.
    Tigges M, Marquez-Lago TT, Stelling J, Fussenegger M (2009) A tunable synthetic mammalian oscillator. Nature 457:309–312. doi: 10.1038/nature07616CrossRefPubMedGoogle Scholar
  3. 3.
    Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403:339–342. doi: 10.1038/35002131CrossRefPubMedGoogle Scholar
  4. 4.
    Siciliano V, Menolascina F, Marucci L et al (2011) Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line. PLoS Comput Biol 7(6):e1002074. doi: 10.1371/journal.pcbi.1002074CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Liu C, Fu X, Liu L et al (2011) Sequential establishment of stripe patterns in an expanding cell population. Science 334:238–241. doi: 10.1126/science.1209042CrossRefPubMedGoogle Scholar
  6. 6.
    Chau AH, Walter JM, Gerardin J et al (2012) Designing synthetic regulatory networks capable of self-organizing cell polarization. Cell 151:320–332. doi: 10.1016/j.cell.2012.08.040CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lienert F, Lohmueller JJ, Garg A, Silver PA (2014) Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nat Rev Mol Cell Biol 15:95–107. doi: 10.1038/nrm3738CrossRefGoogle Scholar
  8. 8.
    Stanton BC, Nielsen A a K, Tamsir A et al (2013) Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat Chem Biol 10:99–105. doi: 10.1038/nchembio.1411CrossRefGoogle Scholar
  9. 9.
    Lohmueller JJ, Armel TZ, Silver PA (2012) A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Nucleic Acids Res 40:5180–5187. doi: 10.1093/nar/gks142CrossRefGoogle Scholar
  10. 10.
    Li Y, Jiang Y, Chen H et al (2015) Modular construction of mammalian gene circuits using TALE transcriptional repressors. Nat Chem Biol 11:207–213. doi: 10.1038/nchembio.1736CrossRefGoogle Scholar
  11. 11.
    Qi LS, Larson MH, L a G et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. doi: 10.1016/j.cell.2013.02.022CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kiani S, Beal J, Ebrahimkhani MR et al (2014) CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat Methods 11:723–726. doi: 10.1038/nmeth.2969CrossRefGoogle Scholar
  13. 13.
    Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. doi: 10.1038/nmeth.1318. nmeth.1318 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Didovyk A, Borek B, Hasty J, Tsimring L (2015) Orthogonal modular gene repression in Escherichia coli using engineered CRISPR/Cas9. ACS Synth Biol 5(1):81–88. doi: 10.1021/acssynbio.5b00147. 150930102638002CrossRefGoogle Scholar
  15. 15.
    Xu Q, Schlabach MR, Hannon GJ, Elledge SJ (2009) Design of 240,000 orthogonal 25mer DNA barcode probes. Proc Natl Acad Sci U S A 106:2289–2294. doi: 10.1073/pnas.0812506106CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Davis JH, Rubin AJ, Sauer RT (2011) Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res 39:1131–1141. doi: 10.1093/nar/gkq810CrossRefPubMedGoogle Scholar
  17. 17.
    Guye P, Li Y, Wroblewska L et al (2013) Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res 41:3–8. doi: 10.1093/nar/gkt605CrossRefGoogle Scholar
  18. 18.
    Torella JP, Boehm CR, Lienert F et al (2014) Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res 42:681–689. doi: 10.1093/nar/gkt860CrossRefGoogle Scholar
  19. 19.
    Casini A, MacDonald JT, De Jonghe J et al (2014) One-pot DNA construction for synthetic biology: the modular overlap-directed assembly with linkers (MODAL) strategy. Nucleic Acids Res 42:e7. doi: 10.1093/nar/gkt915CrossRefGoogle Scholar
  20. 20.
    Casini A, Christodoulou G, Freemont PS et al (2014) R2oDNA designer: computational design of biologically neutral synthetic DNA sequences. ACS Synth Biol 3(8):525–528. doi: 10.1021/sb4001323CrossRefGoogle Scholar
  21. 21.
    Andronescu M, Aguirre-Hernández R, Condon A, Hoos HH (2003) RNAsoft: a suite of RNA secondary structure prediction and design software tools. Nucleic Acids Res 31:3416–3422. doi: 10.1093/nar/gkg612CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zuker M, Mathews DH, Turner DH (1999) Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. RNA Biochem Biotechnol 70:11–43. doi: 10.1007/978-94-011-4485-8CrossRefGoogle Scholar
  24. 24.
    Salis HM (2011) The ribosome binding site calculator. Methods Enzymol 498:19–42. doi: 10.1016/B978-0-12-385120-8.00002-4CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Centre for Synthetic Biology and InnovationImperial CollegeLondonUK
  2. 2.Department of MedicineImperial CollegeLondonUK

Personalised recommendations