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Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides

Nature Methods 10, 647–652 (2013) | Download Citation

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Abstract

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14–378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

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Acknowledgements

Funded by the Swedish Research Council (Vetenskapsrådet) through a repatriation grant and a project grant to B.H. (grants 2010-6296 and 2010-5060). B.H. is a recipient of an assistant professorship with startup funding by Carl Bennet AB, Karolinska Institutet and Vinnova. We thank S. Douglas for help with the 10-helix bundle design, members of the Laboratory of Chemical Biology at Karolinska Institutet for HPLC support, T. Karlberg and members of the Protein Science Facility for crystallography support, M. Schultz and C. Sandén for help with pseudogene cloning and enzymatic reactions for the long ODNs, and N. Seeman for fruitful discussions.

Author information

Affiliations

  1. Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

    • Cosimo Ducani
    • , Corinna Kaul
    •  & Björn Högberg

  2. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Martin Moche

  3. Wyss Institute for Biologically Inspired Engineering at Harvard, Boston, Massachusetts, USA.

    • William M Shih

  4. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • William M Shih

  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • William M Shih

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Contributions

C.D., C.K. and B.H. contributed to experiments. M.M. contributed to the crystallography experiments. B.H. conceived the method principle, and B.H. and W.M.S. contributed to the method design. C.D. and C.K. contributed to method development and implementation. All authors contributed to figure production and manuscript writing.

Competing interests

B.H. is the cofounder and chief executive officer of Basestack Labs AB that will commercialize some applications of the methods presented in this paper.

Corresponding author

Correspondence to Björn Högberg.

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DOI

https://doi.org/10.1038/nmeth.2503