DNA duplex-quadruplex exchange as the basis for a nanomolecular machine

Abstract

There is currently great interest in the design of nanodevices that are capable of performing linear or rotary movements. Protein molecular machines are abundant in biology but it has recently been proposed that nucleic acids could also act as nanomolecular machines in model systems. Several types of movements have been described with DNA machines: rotation and "scissors-like" opening and closing. Here we show a nanomachine that is capable of an extension-contraction movement. The simple and robust device described here is composed of a single 21-base oligonucleotide and relies on a duplex-quadruplex equilibrium that may be fueled by the sequential addition of DNA single strands, generating a DNA duplex as a by-product. The interconversion between two well defined topological states induces a 5-nm two-stroke, linear motor-type movement, which is detected by fluorescence resonance energy transfer spectroscopy.

Figure 1

Presentation of the system. (A) Switching between an intramolecular quadruplex (left) and a duplex (right). An intramolecular quadruplex is formed by the folding of a 21-base oligonucleotide that contains four blocks of three guanines mimicking the vertebrate telomeric motif. The schematic topology proposed here corresponds to the Na⁺ solution structure (19). The K⁺ motif would also lead to a closely similar 5′–3′ distance (18). The 5′ fluorescein and 3′ tetramethylrhodamine groups are depicted by green and orange triangles, respectively. The C-fuel strand is complementary to the F21T sequence, with six extra bases allowing duplex nucleation with the G-fuel strand. (B) Induced movement. Dethreading/rethreading of the DNA device is reminiscent of the movement of a piston in a cylinder. Double-stranded DNA has a persistence length of 100 bp or more (33), indicating that a 21-bp duplex should behave like a rigid rod.

Figure 2

Sequence of the oligonucleotides used in this study. Names are given on the right side. (Top) G-quadruplex forming oligonucleotide. F, fluoresceine; T, tetramethylrhodamine. The 3′ overhang (6–12 bases long) present on the C-fuel strands is shown with lowercase letters. Mismatched bases (with respect to the F21T oligonucleotide) are underlined and boldfaced. The 5′ overhang on the G-fuel strand is shown with lowercase letters. For the 24Gmut control oligonucleotide, this overhang is not complementary to the last six bases of the C-fuel strand and is therefore presented with underlined and bold letters.

Figure 3

The opening step. (Top) Principle of the experiment. Quadruplex-to-duplex conversion is monitored by an increase in fluorescence emission at 520 nm. The 5′ fluorescein and 3′ tetramethylrhodamine groups are depicted by green and orange triangles, respectively. (Middle and Bottom) Effect of temperature on quadruplex-to-duplex conversion. All experiments were performed at four different temperatures in a 10 mM sodium cacodylate pH 7.2 buffer containing 0.1 M LiCl (Middle Left), 0.1 M NaCl (Middle Right), or 0.1 M KCl (Bottom). The predominant starting structure (t = 0 s) before C-fuel strand addition is always an intramolecular quadruplex, except at 45°C in the presence of Li⁺, where F21T is mainly single-stranded.

Figure 4

Optimization of the device. (A) Effect of magnesium. Only one cycle is presented. The closing step is much faster in the presence (solid line) than in the absence (dashed line) of 20 mM MgCl₂. The experiment was performed at 37°C in a 100 mM NaCl/10 mM sodium cacodylate (pH 7.2) buffer. F21T, 33C, and 30G oligonucleotide concentrations were 0.2, 0.25, and 0.25 μM, respectively. (B) Concentration dependency. All experiments were performed at 45°C in a 20 mM MgCl₂/100 mM KCl/10 mM sodium cacodylate (pH 7.2) buffer. On the dotted line, the concentrations for F21T, C-fuel (3′-TGCAATGCCAATCGCAATCGCAATCCC-5′ 27Cm3C), and G-fuel (5′-ACGTTACGGTTAGCGTTAGCGTTA-3′ 24Gm3G) are 0.2, 0.25, and 0.25 μM, respectively. The full line presents a similar experiment with a 10-fold increase in all oligonucleotide concentrations (normalized fluorescence intensity).

Figure 5

Cycling the device. (A) With DNA strands. By alternatively adding stoichiometric amounts of the C-fuel (27Cm3C; 2.5 μM) and G-fuel (24Gm3G; 2.5 μM) strands, F21T may be opened and closed repeatedly (at least 11 times); for purposes of clarity, the times of C- and G-fuel additions are indicated for only one cycle. Each addition of the C- or G-fuel strand results in a 0.5% dilution of the reactants, which is mathematically corrected on this graph. Experimental conditions: 100 mM KCl/20 mM MgCl₂/10 mM sodium cacodylate (pH 7.2) at 45°C. η, the average cycling efficiency (3% loss per cycle). (B) Cycling with a modified oligonucleotide. The G-fuel strand is a chemically modified oligonucleotide (morpholino, with a neutral backbone, synthesized by Gene Tools). Nine successive cycles are shown. Despite a careful assessment of strand concentrations, each successive cycle leads to a significant loss in signal. The experiment was performed in a 100 mM KCl/10 mM sodium cacodylate (pH 7.2) buffer at 45°C (no magnesium). Oligonucleotide sequences are shown in C.