Circular dichroism as a means to follow DNA gymnastics : on the shoulders of giants

Introduction Many of the key goals of DNA-based nanotechnology entail the use of periodic arrays with tunable features such as binding patterns and cavities, with important applications in medicine as drug-delivery systems. Branched networks that result from ligating three-way junctions of DNA together have been reported. This study introduces a new method to build either linear or three-dimensional self-assembling structures. Pairs of DNA stem-loops (46 bases) were designed to self-assemble by foot-loop interactions to form networks of strings (A+B) or spirals (C+D) (Fig. 1). The inter-subunit interactions are restricted to cooperative Watson-Crick hydrogen bonds. No ligation is used—making the assembly completely reversible.


Introduction
Many of the key goals of DNA-based nanotechnology entail the use of periodic arrays with tunable features such as binding patterns and cavities, 1 with important applications in medicine as drug-delivery systems. 2 Branched networks that result from ligating three-way junctions of DNA together have been reported. 3,4This study introduces a new method to build either linear or three-dimensional self-assembling structures.Pairs of DNA stem-loops (46 bases) were designed to self-assemble by foot-loop interactions to form networks of strings (A+B) or spirals (C+D) (Fig. 1).The inter-subunit interactions are restricted to cooperative Watson-Crick hydrogen bonds.No ligation is used-making the assembly completely reversible.

Oligonucleotide design and synthesis
The sequences were designed to form a double-stranded stem of alternating guanine/cytosine base pairs 5 holding a singlestranded, asymmetrical loop sequence from the polypurine tract of HIV-1 (A 4 GA 4 G 6 A). 6 The 'feet' (5'-and 3' extensions of the stem) were added to make complementary base pairs to the loop, thus forming either strings or spirals.Heterodimers were required to make the foot-loop interaction constitute an antiparallel double helix.Oligonucleotides were synthesised by standard phosphoramidate chemistry using a Beckman 1000 M DNA synthesiser, and purified in dimethoxytrityl-on mode by reverse-phase high performance liquid chromatography (HPLC) using an acetonitrile gradient.Concentrations were expressed in strand molarity, using nearest-neighbour approximation for the extinction coefficients of the unfolded species. 7

Ultraviolet (UV) melting
Thermal melting curves were recorded using a heating rate of 1°C min -1 in an Uvikon spectropolarimeter, with a custom-made heating block equipped with an Oasis analog-to-digital converter.The DNA strand concentration was 1.5 µM ml -1 and oligonucleotides were dissolved in 100 mM l -1 sodium chloride, 5 mM l -1 magnesium chloride, and 10 mM l -1 sodium cacodylate, at pH 7.0. 8

Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy of 1.5 µM ml -1 DNA strand concentration in 100 mM l -1 sodium chloride, 5 mM l -1 magnesium chloride, and 10 mM l -1 sodium cacodylate, at pH 7.0 in a 1 ml jacketed quartz cuvette, was performed in a Jasco J-810 spectropolarimeter with ten accumulations per scan.Circular dichroism, with units of millidegrees, reflects detailed helical  geometry and is defined as the difference between the absorption of left-and right-handed, circularly-polarised light measured as a function of wavelength (nm).The sample was kept at a constant temperature by a Haake D8 programmable water bath during each set of scans.

Polyacrylamide gel electrophoresis (PAGE)
Native PAGE was performed with samples at a DNA strand concentration of 1 µM ml -1 , pre-heated to 80°C, and incubated at room temperature in 100 mM l -1 sodium chloride, 5 mM l -1 magnesium chloride, and 10 mM l -1 sodium cacodylate, pH 7.0, before loading on a 10% gel run at 70 volts with tris-borate running buffer (pH 8.3), and visualised under UV light by ethidium bromide intercalation.

Melting behaviour of the networks
The melting of assemblies (Fig. 1.) was biphasic at a neutral pH (Fig. 2.) corresponding to the denaturation of the intermolecular 'foot-loop' double helix (melting temperature (Tm) = 52°C), followed by the unfolding of the intramolecular stem double helix (Tm = 82°C) in 1 ml of 100 mM l -1 sodium chloride, 5 mM l -1 magnesium chloride, and 10 mM l -1 sodium cacodylate.

Circular dichroism
Circular dichroism spectra revealed that the network made up of oligomers C+D showed more helicity (15 m°) than oligomers A+B (13 m°) at 272 nm (Fig. 3a), as expected.Upon raising the temperature above the melting temperature (Tm, the temperature at which half of the observed absorbance change has been recorded) of the 'foot-loop' interactions, the spectra of the individual stem-loops were similar at 70°C (Fig. 3b).Further heating to 90°C resulted in similar spectra for the random coil species (Fig. 3c).

Polyacrylamide gel electrophoresis
Lanes 1, 2 and 3 of Fig. 4 show the monomer stem-loop D and the dimers and higher networks formed by A+B and C+D, respectively.The migration patterns of lanes 2 and 3 were different, as expected, supporting the CD evidence that networks A+B and C+D had different secondary structures.

Conclusion
This is the first time that 'foot-loop' interactions have been shown to create either linear strings or spirals that were distinguished by CD.Future work will include electron and atomicforce microscopy of the networks, as well as incorporate a transcription factor-binding site into the stem sequence.Hetero-multimers, whether they are formed by single strands or