A new building block for DNA network formation by self-assembly and polymerase chain reaction

Abstract

The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

Keywords: AFM; DNA; DNA polymerase; PCR; branched DNA; nanotechnology; nucleic acids; self-assembly.

Scheme 1

Reagents and conditions: (a) PdCl₂(PPh₃)₂, DMF, CuI, NEt₃, 55 °C, microwave, 82%; (b) PdCl₂(PPh₃)₂, DMF, CuI, NEt₃, 80 °C, microwave, 48%; (c) CH₂Cl₂, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine, 0 °C, 84%.

Scheme 2

Stepwise solid-phase synthesis for branched oligonucleotides. (I): The first oligonucleotide branch is synthesized in 3'–5' direction using standard phosphoramidites. (II): Incorporation of the branching point by usage of 6. (III): Simultaneous synthesis of the two remaining oligonucleotide branches in 5'–3' direction using inverse protected phosphoramidites.

Figure 1

(A) Depiction of synthesized branched oligonucleotides; (B) Sequences of all synthesized branched oligonucleotides. Bp: branchpoint.

Figure 2

Thermal denaturating studies of self-complementary branched oligonucleotides. Different conditions are indicated in the figure. TEAA: triethylammonium acetate.

Figure 3

Generation of DNA networks with Taq DNA polymerase from 1062 nt template using primer ODN I and ODN II. Monitoring of DNA network growth influenced by varying of annealing temperature (as indicated) and increasing cycles (10, 18, 22, 28, respectively).

Figure 4

AFM analysis of DNA structures: (A) Overview-scan 10 × 10 µm showing DNA networks generated after 28 cycles with 66 °C annealing temperature (bar is 2 µm). (B, C) Zoom in picture of DNA network (all bars are 0.5 µm, respectively). (D) AFM when non-branched primers were employed (bar is 0.5 µm). (E) Software analysis of AFM measurement taking first derivative of height channel. DNA network generated after 28 cycles with 66 °C annealing temperature (bar is 0.5 µm).

Figure 5

EPR spectra with corresponding spectral simulations (red line) of (A) dT*TP, (B) PCR reaction product resulting from non-branched primers in the presence of 50% dT*TP; The simulated spectrum and the corresponding τ_C are derived from a one component fit, (C) DNA networks generated by PCR in the presence of 50% dTTP* using branched primer; the simulated spectrum and corresponding τ_C are derived from a two component fit, (D) structure of modified dT*TP employed in PCR.