Design and Characterization of Programmable DNA Nanotubes

Abstract

MethodsDesign, data-processing and modelling: DNA sequences were designed using our own algorithms based on sequence symmetry minimization implemented in Matlab and C (available at http://www.dna.caltech.edu/DNAdesign/). Curve fits for persistence length data and models for lattice strain energies were calculated in Matlab.Molecular models were constructed and visualized using a combination of NAMOT, RasMol, and PyMol (scripts and coordinates at http://www.dna.caltech.edu/SupplementaryMaterial/).The molecular models are for visualization only and have not been subjected to molecular dynamics calculations.DNA sample preparation: Lyophilized HPLC-or PAGEpurified DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA), resuspended in water, quantitated by UV absorbance at 260 nm, and stored at -20• C. All samples were prepared in a 1X Tris-Acetate-EDTA (TAE) buffer with 12.5 mM magnesium acetate (pH=8.3). An equimolar mixture of strands (5 strands if one tile, 10 strands if two) was annealed from 95• C to 25• C (fluorescence microscopy) steps in a PCR machine (Eppendorf Mastercycler). For AFM, each strand was present at 200 nM, for fluorescence microscopy the total concentration of tiles was kept at 400 nM. For fluorescence microscopy, a single fluorescein-labeled strand was incorporated into each tile; the position of the dye was varied from the 5 end of the #3 strand to the 5 end of the #5 strand with no apparent effect. AFM of REp+SEp(3:FAM) was similar to that of REp+SEp.Preparation of PVP coated glass: Adapted from.1 Microscope slides and coverslips were washed in 1M NaOH for 1 hour, rinsed thoroughly with de-ionized (DI) water and immersed in 1% v/v acetic acid solution for 2 hours. Then, they were rinsed again with DI water and silanized in a 1% v/v 3-(trimethoxysilyl)propylmethacrylate (Aldrich) in 1% v/v acetic acid for 36 hours. For polymer coating, 500 mL of a 4% w/v Mw = 360, 000 poly(vinylpyrrolidone) (PVP, USB Corp.) solution with 2.5 mL of 10% w/w ammonium persulfate solution and 250 µl of N,N,N ,N -tetramethylethylenediamine (TEMED, Acros) was prepared. Slides and coverslips were incubated in the PVP solution at 80• C for 18 hours. They were then rinsed and stored in DI water. Coating was stable for at least 2 weeks.Preparation for fluorescence microscopy: Samples were left overnight at room temperature after annealing. Immediately prior to use, a PVP-coated microscope slide and coverslip were rinsed with ethanol and dried. Then, 2.6 µl of solution containing DNA tubes and oxygen scavenging system (0.035 mg/ml catalase, 0.2 mg/ml glucose oxidase, 4.5 mg/ml glucose, 5% β-mercaptoethanol) was deposited onto the slide, covered with the coverslip and sealed with epoxy or parafin. The distance between slide and coverslip was ≈5 µm and the thickness of sample solution was typically narrowed to ≈3 µm by the PVP coating.Fluorescence microscopy: Samples were imaged on an inverted microscope (IX 70, Olympus) with 100X/1.40 NA oil immersion and 40X/0.75 NA air objectives. Blue lig...

Sturdier DNA Nanotubes via Ligation

“…3 These persistence lengths are several times longer than those observed previously. 3 Here we used higher DNA concentrations and a slower thermal anneal, both of which should reduce the occurrence of lattice defects and favor larger tube diameters. The observation that ligation does not noticeably influence stiffness at room temperature reinforces the notion that lattice structure, rather than sticky end stability, is the primary determinant of nanotube stiffness.…”

“…[1][2][3] This combination of structural and biochemical properties makes DNA nanotubes especially interesting for applications. For example, they might be metallized or functionalized by surface attachment of biomolecules or nanoparticles and so serve as interconnects in self-assembled networks.…”

“…The latter suggestion is supported by high-resolution AFM images of tile lattices on mica that show relatively wide separations (with an apparent width of 2-3 nanometers) in these regions. 3 The core nicks, by contrast, each lie on a region of helix connected to one of its neighbors by crossovers only two helical turns apart. High-resolution AFM shows the separation between helices in this region to be <0.5 nm.…”

Two Computational Primitives for Algorithmic Self-Assembly: Copying and Counting

“…This results in the set of four DAE-E molecules shown in Figure 1c. (Although this doublecrossover motif has been shown previously to have intrinsic curvature that encourages assemblies of tiles to roll up into tubes, 17,34 assemblies grown from long scaffolds in that work usually contained 5 to 15 layers of rule tiles, which we predicted would be sufficient for our investigations here.) To distinguish tiles representing 0's from tiles representing 1's, we decorated the latter with protruding hairpin motifs that provide topographic contrast when imaged by atomic force microscopy (AFM).…”

“…buffer on freshly cleaved mica for AFM imaging using fluid tapping mode (as described for counting "tube falling events" in ref 34). During AFM imaging, after landing on the mica, tubes first appear as thick indistinct linear objects (closed tubes) and subsequently pop open to lie flat on the mica such that individual tiles can be resolved.…”