Facile and scalable preparation of pure and dense DNA origami solutions

Abstract

DNA has become a prime material for assembling complex three-dimensional objects that promise utility in various areas of application. However, achieving user-defined goals with DNA objects has been hampered by the difficulty to prepare them at arbitrary concentrations and in user-defined solution conditions. Here, we describe a method that solves this problem. The method is based on poly(ethylene glycol)-induced depletion of species with high molecular weight. We demonstrate that our method is applicable to a wide spectrum of DNA shapes and that it achieves excellent recovery yields of target objects up to 97 %, while providing efficient separation from non-integrated DNA strands. DNA objects may be prepared at concentrations up to the limit of solubility, including the possibility for bringing DNA objects into a solid phase. Due to the fidelity and simplicity of our method we anticipate that it will help to catalyze the development of new types of applications that use self-assembled DNA objects.

Keywords: DNA nanotechnology; DNA origami; molecular crowding; poly(ethylene glycol); self-assembly.

Figure 1

PEG purification of a library of DNA origami objects. a) Illustration (RR to 100 hb) and CanDo-computed²⁰ models (plate1 to 42 hb_b) of a panel of DNA objects comprising a variant of Rothemund’s single-layer DNA rectangle (RR) and five multilayer objects (four-, ten-, 18-, or 42-helix bundle in honeycomb lattice design; 100-helix bundle in square lattice design), a plate-like object with aperture and double-stranded loop (plate1), a plate-like object (plate2), an asymmetric 82-helix bundle in square lattice design (pointer), a gear-like object (gear), a flexible hinged-bar object (hinge), a letter-A-like object (“A”), a straight and bent version of a robot-like object (robot, robot_b),²⁰ a letter-S-like object (“S”), a bow-like object (bow), and bent versions of an 18-helix bundle and a 42-helix bundle (18hb_b, 42hb_b)., The library of objects thus samples a wide spectrum of shapes, aspect ratios, and mechanical properties. b) Images of agarose gels on which unpurified self-assembly reaction mixtures (U) and PEG purified samples (P) of the objects listed above in (a) were electrophoretically separated. Labels: po, gel loading pocket; m, properly folded monomers; ex, non-integrated excess staple strands. c) Exemplary TEM micrographs of single particles in unpurified reaction mixtures (U) and PEG-purified samples (P). Scale bars: 50 nm.

Figure 2

Agarose-gel-electrophoretic characterization of PEG purification using exemplarily a 42-helix bundle object. a) Image of a gel on which samples extracted after each of ten consecutive PEG purification cycles (1–10) were separated. Labels: sc, reference sample containing only scaffold strands; U, unpurified self-assembly reaction mixture; po, gel loading pocket; m, folded objects; ex, non-integrated excess staple strands. b) Cross-sectional lane profiles determined from (a). c) Recovery of folded objects (left) and residuals of excess staple strands (right) relative to unpurified reaction mixture, as determined by integrating and comparing the areas of the peaks reflecting folded objects and excess strands, respectively. The experiment was run in triplicate, each experiment gave data as in (a). Error bars in (c) indicate the standard deviation in the recovery and residuals, respectively. d) Image of a gel containing samples extracted after one and five cycles of PEG purification (1xP, 5xP), after one and five cycles of molecular-weight cut-off filtration (1xF, 5xF), after AGE extraction (1xG), and after PEG purification of a previously AGE-extracted sample (G+P). U refers to the unpurified self-assembly reaction mixture. Other labels as in (a). e) Cross-sectional lane profiles determined from (d). f) Recovery of folded objects and residual excess strands as in (c) but for samples as in (d). Values were obtained from three independent experiments, each giving data as in (d). Error bars indicate the standard deviation in the recovery and residuals, respectively. g–i) Estimation of residual PEG content in purified samples using fluorescein-labeled PEG (fPEG). g) Overlay image of two scans of the same gel, recorded separately for the ethidium bromide and fluorescein emission channels. Samples were taken before (U) and after (U+PB) addition of precipitation buffer (PB) and compared to the supernatant (SN) and the redissolved pellet (P) of the precipitation. h) Cross-sectional lane profiles from ethidium bromide channel (dark gray) and fluorescein channel (light gray). i) Estimated concentrations of folded DNA objects (dsDNA) and PEG at all steps of a PEG purification cycle. Labels: fPEG, fluorescein-labeled PEG; other labels as in (a).

Figure 3

Preparation of solid material and dense solutions containing intact DNA objects. a) Approximately 6.8 mg of solid material containing a variant of Rothemund′s rectangle (unlabeled). b) Approximately 5.5 mg of solid material containing 42-helix bundle objects, labeled each with one ATTO655 dye. c) Approximately 8.4 mg of solid material containing 24-helix bundle objects, labeled each with ten cyanine-3 dyes on average. d) Left: unpurified self-assembly reaction mixture containing approximately 50 nm of folded 24-helix bundle objects and 150 nm excess staple strands; center: dense solution containing approximately 5.6 μm of folded 24-helix bundle objects, prepared by redissolving the materials from (c); right: reference sample containing 70 μm of a cyanine-3 modified DNA oligonucleotide. e–g) TEM micrographs of dried and redissolved single-layer rectangle (e), 42-helix bundle (f), and 24-helix bundle (g); insets: average single-particle micrographs obtained from non-dried (left) and dried and redissolved samples (right). Scale bars: 50 nm (field-of-view micrographs), 10 nm (insets).