NANO LETTERS
Cationic Comb-Type Copolymers for Boosting DNA-Fueled Nanomachines
2007 Vol. 7, No. 1 172-178
Sung Won Choi,†,‡ Naoki Makita,§,‡ Satoru Inoue,§ Charles Lesoil,† Asako Yamayoshi,† Arihiro Kano,† Toshihiro Akaike,§ and Atsushi Maruyama*,†,| Institute for Materials Chemistry and Engineering, Kyushu UniVersity, 6-10-1 Hakozaki, Higashi, Fukuoka 812-8581, Japan, Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama 226-8501, Japan, and CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Received November 9, 2006
ABSTRACT For the better applications and developments of DNA nanomachines, their responding kinetics, output, and sequence-selectivity need to be improved. Furthermore, the DNA nanomachines currently have several limitations in operating conditions. Here we show that a simple addition of a cationic comb-type copolymer, poly(L-lysine)-graft-dextran, produces the robust and quick responses of DNA nanomachines under moderate conditions including physiologically relevant conditions even at very low strand concentrations (nanomoles per liter range) through hybrid stabilization and DNA strand exchange acceleration.
Recently, applications of DNA to nanotechnology fields have attracted increasing interest because of its sequence-specific interaction and robust physicochemical nature. Various DNA molecular constructs, such as polyhedral crystals 1-3 and twodimensional DNA arrays4 have been reported. Yurke et al. first demonstrated a DNA-fueled nanomachine which undergoes the structural transition from one to another in response to added DNA fuels.5 Thereafter several types of DNA nanomachines exhibiting unique actions have been reported.6-9 DNA nanomachines that operate in response to pH change10 or particular substrates,11-13 through combination with DNA enzyme, have also been described. The controlled activation and release of proteins such as thrombin14 and coagulation factor IXa15 by using nanomachines based on RNA or DNA aptamers have been recently demonstrated, where the activities of the proteins are regulated by oligonucleotides complementary to the aptamers. These studies implied the promising applications of the nanomachines in bionanotechnology fields. The common operating principles employed in the DNAfueled nanomachines involve a couple of sequence-specific reactions between DNAs. One is hybridization between a set of complementary single-stranded (ss) DNAs, and the other is strand exchange reaction between a double-stranded * To whom correspondence may be addressed. Tel: +81-92-642-3097. Fax: +81-92-642-4224. E-mail:
[email protected]. † Kyushu University. ‡ These authors contributed equally to this work. § Tokyo Institute of Technology. | Japan Science and Technology Agency. 10.1021/nl0626232 CCC: $37.00 Published on Web 12/08/2006
© 2007 American Chemical Society
(ds) DNA hybrid and its complementary ssDNA. In the strand exchange reaction, a DNA strand in the preformed hybrid is replaced by the ssDNA. There are at least three requirements for better developments and applications of the DNA nanomachines. First, the output that can be produced by the machines must be enhanced. To this end, the frame strength of the machine bodies should be also improved. Second, the quickness of machine responses should be secured. Third, the sequence selectivity of the machines needs to be increased.16 The output and frame strength of the machines depend on the stability of DNA hybrids, while the quick response relies on hybridization and, more specifically, strand exchange reaction rates. It is difficult to satisfy both requirements simultaneously because of the dilemma that an increase in hybrid stability generally results in a decrease in the strand exchange rates. For acquiring rapid and robust actions, the DNA nanomachines currently have several limitations in DNA strands and operating conditions such as DNA strand concentration, temperature, and salt conditions.5,7,17,18 For example, faster kinetics could not be achieved without increasing strand concentration up to micromoles per liter range.7,17,18 Operating temperature was also raised to near the melting point (Tm) of the machine to obtain quick responses.17,18 However, this should bring about the loss of the frame strength and output of the machine. Sequence and chemical modifications of fuel strands were also employed to accelerate the strand exchange reaction rate by modulating the stability of the machine’s hybrids.17, 18
Figure 1. Materials used in this report. (A) Structural formula of poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer. (B) Operating diagram and strand sequences of a DNA-fueled “chameleon tongue” nanomachine proposed by Mergny et al.17
These facts explain well that it is difficult to design functional molecular devices through combining various nanomachines. Thus, a novel concept to resolve the dilemma is indispensable for the further development of this field. We have studied interactions between DNA and cationic comb-type copolymers, poly(L-lysine)-graft-dextran (PLLg-Dex, Figure 1A), which have abundant water-soluble side chains (>80 wt %).19,20 We showed that the copolymer does not induce coil-globule transition of DNA and forms a totally soluble inter-polyelectrolyte complex with DNA.21 Interestingly, the copolymers considerably accelerate DNA strand exchange reaction while stabilizing DNA hybrids.22,23 The strand exchange reaction accelerated by the copolymer was demonstrated to be highly sequence-sensitive, permitting us to distinguish single-base mismatches quickly and precisely.24 Thus, these properties of the copolymer are considered to be suitable to overcome the dilemma underlying the DNA nanomachines. Here we show that a simple addition of PLL-g-Dex considerably improves the responses and robustness of the DNA nanomachines. Moreover, the copolymer allows the machine’s dynamic action without carefully adjusted conditions even at about 200 times lower strand concentration (nanomole per liter range) than those in previous reports.7,17,18 To our knowledge, the copolymer is the first example of an adjuvant material that improves DNA nanomachine responses. We first investigated the effect of PLL-g-Dex on the performance of a “chameleon tongue” DNA nanomachine proposed by Mergny et al.17 (Figure 1B). In the folded state, the machine “Body” forms an intramolecular G-quadruplex. Upon an addition of “Fuel 1 (F1)”, the Body transforms into the extended state through duplex formation with F1. The successive addition of “Fuel 2 (F2)” removes F1 from the Nano Lett., Vol. 7, No. 1, 2007
Figure 2. Transformation of the nanomachine in the absence or presence of PLL-g-Dex. (A) Native-PAGE of Body strand and its mixture with F1 and F2 strands with (left panel) or without annealing treatment (right panel): lane M, 20-mer ssDNA marker (5′-TAC CAC TCG TTC CCG CTC CT-3′); lanes 1-3, samples without PLL-g-Dex (lane 1, Body alone; lane 2, equimolar mixture of Body and F1; lane 3, equimolar mixture of Body, F1 and F2). Lanes 4-6 contain the same samples as lanes 1-3 but with PLLg-Dex, keeping copolymer/DNA charge (N/P) ratio constant at a value of 2. Lanes 7-12 contain the same samples as lanes 1-6 without annealing treatment. (B) CD spectra of the Body strand at 25 °C in 10 mM sodium phosphate buffer (pH 7.2) containing 150 mM NaCl and 0.5 mM EDTA in the absence (solid line) or presence (dashed line) of PLL-g-Dex.
Body/F1 duplex through strand exchange reaction to reset the Body strand to the G-quadruplex-folded state. PAGE assays were performed with or without annealing treatment (Figure 2A) to check the proper transformations of the machine in response to the added fuel DNAs. In the absence of the copolymer, the Body (lanes 1 and 7) adopted a compact intramolecular G-quadruplex which was featured by faster electrophoretic migration than the ssDNA marker (lane M). Transformation of the Body to the extended state due to the duplex formation with F1 was confirmed by the mobility shift (lanes 2 and 8). Furthermore, strand exchange reaction between the Body/F1 duplex and F2 was confirmed by the re-formation of the intramolecular quadruplex (lanes 3 and 9). As shown in lanes 4-6 and 10-12 in Figure 2A, similar results were observed even in the presence of the copolymer. These results indicate that the addition of the copolymer does not influence the transformations of the machine regardless of the annealing treatment. The quadruplex structures were also confirmed by circular dichroism (CD) spectroscopy either in the absence or in the presence of the copolymer (Figure 2B). DNA G-quadruplexes are known to exhibit characteristic profiles of circular 173
Figure 3. Effect of NaCl concentration on the operation of the nanomachine. The experiment was performed at 37 °C in 10 mM sodium phosphate buffer (pH 7.2, 0.5 mM EDTA) containing 50 mM (blue), 150 mM (green), 500 mM (orange), or 1 M (red) NaCl. F1 and F2 of an equimolar amount to Body (9.6 nM) were added at t ) 0 and t ) 70 min, respectively. The relative fluorescence intensity ) FIt/FI0, where FI0 and FIt represent the fluorescence intensities at the initial state before adding F1 and at time t, respectively.
dichroism with 295 nm positive and 265 nm negative bands.25 Although the concentrations adopted in the experiments are considerably different (see Supporting Information), this result implies that the copolymer might not affect the quadruplex structure and transformations of the machine. We then carried out fluorescence resonance energy transfer (FRET) assay17 for the real-time observation of the nanomachine’s transformation. For FRET assay, the Body strand labeled with both FITC and TAMRA (Figure 1B) was used. In the folded state, the fluorescence emission from FITC is quenched by TAMRA that is situated in the proximity of the FITC. The FITC emission is recovered as the machine turns to the extended state. The effect of ionic strength on the operation of the machine was examined under four different salt conditions ([NaCl] ) 50 mM, 150 mM, 500 mM, or 1 M) (Figure 3). In a previous report, increasing Body strand concentrations from 0.2 to 2 µM with a 1.25 times excess amount of Fuel strands resulted in faster transformation kinetics.17 In this study, however, we adjusted the concentration of Body to 9.6 nM (ca. 200 times lower than that described in the previous report) in order to open the applicability of the machine in the nanomolar range. Moreover, F1 and F2 of the equimolar amount to Body were used. As shown in Figure 3, the operation of the machine was considerably slower at 50 mM NaCl. It took more than 30 and 70 min, respectively, to reach equilibrium state in extending and shrinking steps. Although the response of the machine improved with increasing [NaCl] from 50 to 150 mM, the extending and shrinking steps, respectively, were considerable slower (
