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


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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Figure 1: Outline of the MOSIC ODN production method.
Figure 2: Digestion of the amplified crystal pseudogene releases the correct length ODNs.
Figure 3: Comparison of synthetic, commercial DNA with MOSIC ODNs for crystallization.
Figure 4: A DNA crystal produced with MOSIC ODNs.
Figure 5: DNA origami using MOSIC ODNs.
Figure 6: Applying the MOSIC method for production of very long DNA oligonucleotides.

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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.

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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.

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Correspondence to Björn Högberg.

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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.

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Supplementary Figures 1–17, Supplementary Tables 1–3, Supplementary Note 1 (PDF 4746 kb)

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Ducani, C., Kaul, C., Moche, M. et al. Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides. Nat Methods 10, 647–652 (2013).

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