Synapsable quadruplex-mediated fibers

Abstract

We have fabricated a DNA-based nanofiber created by self-assembly of guanine quadruplex (Hoogsteen base pairing) and double-stranded DNA (Watson-Crick base pairing). When duplexes containing a long stretch of contiguous guanines and single-stranded overhangs are incubated in potassium-containing buffer, the preformed duplexes create high molecular weight species that contain quadruplexes. In addition to observation of these larger species by gel electrophoresis, solutions were analyzed by atomic force microscopy to reveal nanofibers. Analysis of the atomic force microscopy images indicates that fibers form with lengths ranging from 250 to 2,000 nm and heights from 0.45 to 4.0 nm. This work is a first step toward the creation of new structurally heterogeneous (quadruplex/duplex), yet controllable, DNA-based materials exhibiting novel properties suitable for a diverse array of nanotechnology applications.

Figure 1

Duplex precursor assembly in TMACl assessed by native PAGE. Lane 1, 4.0 × 10⁻⁵ mol/L (40 μM) SQ1A:SQ1B duplex; lane 2, mixture of 4.0 × 10⁻⁵ mol/L (40 μM) C1A:C1B duplex and 8.0 × 10⁻⁵ mol/L (80 μM) single-stranded C1A. C1A:C1B is a 39-mer blunt-end duplex used as a control. SQ1A:SQ1B is the 39-mer synapsable duplex with overhangs. Gel with a mass fraction of 12% acrylamide was run in 0.01 TMgTB buffer and imaged by UV shadowing.

Figure 2

Native PAGE showing higher order species formed by SQ1A:SQ1B duplex incubated in potassium-containing buffer. Left: Sample concentrations are 1.0 × 10⁻⁴ mol/L (100 μM) per strand SQ1A or SQ1B, 5.0 × 10⁻⁵ mol/L (50 μM) SQ1A:SQ1B duplex, and 5.0 × 10⁻⁵ mol/L (50 μM) C1A:C1B duplex. Gel (acrylamide mass fraction 12%) was run in 0.01 KMgTB buffer and then UV-shadowed. Right: Sample concentrations are 2.0 × 10⁻⁶ mol/L (2 μM) strand C2, 2.0 × 10⁻⁶ mol/L (2 μM) strand SQ1A, 1.0 × 10⁻⁶ mol/L (1 μM) duplex C2:SQ1A, and 1.0 × 10⁻⁶ mol/L (1 μM) duplex SQ1A:SQ1B. Gel (acrylamide mass fraction 15%) was run in 0.01 KMgTB buffer and then stained with Sybr Green I dye.

Figure 3

Native gel electrophoresis images showing that quadruplex is present in synapsed (SQ1A:SQ1B)₂. TMACl (top row): Samples in lanes 2, 4, and 6 contain 1.0 × 10⁻⁵ mol/L (10 μM) NMM. Lanes 1 and 2, 4.0 × 10⁻⁵ mol/L (40 μM) SQ1A:SQ1B duplex; lanes 3 and 4, mixture of 4.0 × 10⁻⁵ mol/L (40 μM) C1A:C1B duplex with 1.0 × 10⁻⁴ (100 μM) C1A; lanes 5 and 6, 8.0 × 10⁻⁵ mol/L (80 μM) per strand SQ1A. Gel (acrylamide mass fraction 12%) was run in 0.01 TMgTB buffer and (a) UV-shadowed (b) or UV-transilluminated. KCl (bottom row): All samples contain 1.0 × 10⁻⁵ mol/L (10 μM) NMM. Lane 1, 4.0 × 10⁻⁵ mol/L (40 μM) C1A:C1B duplex; lane 2, 4.0 × 10⁻⁵ mol/L (40 μM) SQ1A:SQ1B duplex in TMACl; lane 3, 3.0 × 10⁻⁵ mol/L (30 μM) SQ1A:SQ1B duplex incubated overnight at 4°C in high potassium-containing buffer to assemble quadruplex; lane 4, 6.0 × 10⁻⁵ mol/L (60 μM) per strand SQ1A. Gel (acrylamide mass fraction 12%) was run in 0.01 KMgTB buffer and (c) UV-shadowed or (d) UV-transilluminated.

Figure 4

AFM images of the (SQ1A:SQ1B)₂nanofiber. Left panel: The synapsable DNA nanofiber was prepared by dilution of purified SQ1A:SQ1B duplex originally diluted from 0.05 mol/L (50 mM) TMACl into 1 KMgTB buffer. The quadruplex sample was incubated for 12 h at 4°C prior to depositing it on the silicon wafer for imaging. The average height of the nanofiber is 0.45 ± 0.04 nm. Right panel: Gel-purified SQ1A:SQ1B duplex was heated to 90°C for 5 min and kept at 50°C for 72 h. The concentration was 6.7 × 10⁻⁹ mol/L (6.7 nM) quadruplex. A drop of sample was placed on the silicon wafer substrate, evaporated for 10 min at room temperature, and then washed with purified water three times prior to drying at room temperature for 1 to 2 h. Average height above the background of the bundles is 0.9 ± 0.4 nm.

Figure 5

Proposed model for assembly of quadruplex nanofibers.