DNA Nanomachines - and Applications of Smart and

ABSTRACT

5. DNA Nanomachines

Nanomachines, or molecular machines, can be regarded as nanosize-devices that convert energy into mechanical tasks. As the most famous example, and an essential part of life, ATP-powered motor protein is a class of natural nanomachine that can perform mechanical movements in nanoscale. Likely, the conformational switch-ability of DNA also enables the fabrication of nanoscale molecular machines. From the structural point of view, DNA nanomachines are made up of assembled DNA structures which contain both rigid and switchable parts. The switchable part is responsive to external stimuli, such as “fuel” DNA strands and environmental changes, and therefore can generate force and perform movements. In this section we will summarize present efforts on constructions of DNA nanomachines based on different driven mechanisms and further discuss their important applications and future development directions.

5.1 Pr ototypes

Up until now all established DNA nanomachines could be sorted into several catalogues by their original driven mechanisms. The most commonly used one can be considered as the “fuel-strands” strategy. It is well known that a short DNA strand could be replaced by a longer strand to form a more stable duplex, which is called “chain-exchange reaction”

or “strand-exchange reaction”. This reaction was fi rstly employed by Yurke et al. in 2000 (Yurke et al. 2000) to induce motions to DNA-based nanostructures. The device is assembled by three single-strands which can form two rigid duplex arms connected by a hinge section and two dangling ends linked to arms, resembling a pair of tweezers (Fig. 7a).

Upon adding “fuel” and “anti-fuel” strands alternately, the tweezers can be switched between “open” and “closed” states. This fuel-strands

strategy has also been employed to drive other kinds of DNA assemblies to move, including PX-JX₂ complex (Yan et al. 2002), G-quadruplex (Fig. 7b) (Li and Tan 2002), and DNA origami-based nanomachines. In 2010, Seeman et al. demonstrated that a nanoscale assembly line can be realized by the judicious combination of three known DNA-based modules (Gu et al. 2010).

In the meantime, Yan and co-workers developed molecular robots can walk along the prescriptive landscapes, which can also autonomously carry out sequences of actions such as ‘start’, ‘follow’, ‘turn’ and ‘stop’ (Lund et al.

2010). In 2011, Turberfi eld and co-workers assembled a 100-nm-long DNA track on a two-dimensional scaffold, and observed stepwise movement of a synthetic molecular transporter directly by AFM (Wickham et al. 2011).

The next year, they achieved another DNA-based molecular motor that can navigate a network of tracks (Wickham et al. 2012).

(b) A G-quadruplex-based DNA nanomachine. Reprinted with permission from ref. (Li and Tan 2002). Copyright 2002 American Chemical Society. (c) An i-motif-based DNA nanomachine which is responsive to pH change. Reprinted with permission from ref. (Liu and Balasubramanian 2003). Copyright 2003 John Wiley & Sons Ltd. (d) A light-driven i-motif- based DNA nanomachine. Reprinted with permission from ref. (Liu et al. 2007). Copyright 2007 John Wiley & Sons Ltd.

In principle, the above fuel-strands strategy could be applied to all strand-exchange reaction-powered DNA nanomachines, since, as we have mentioned, hybridization is the common feature of DNA. However, the main disadvantage is these reactions will result in cumulated duplex wastes.

These useless duplexes may compete with surrounding nanomachines and, from the point of entropy fl ow, the accumulation of waste DNA will increase the entropy of the system, making the machine dead in fi nal.

To avoid duplex wastes, non-DNA stimuli should also be choices for controlling motions. In fact, this approach has already been proposed in the construction of the fi rst DNA-based nanomechanical device (Mao et al. 1999), in which case ethidium ions were used as intercalators to induce branch point migration in a tetramobile branched junction structure.

G-quadruplex, in some cases (Fahlman et al. 2003, Miyoshi et al. 2007, Monchaud et al. 2008), can also be responsive to environmental changes and can be switched by metal ions.

However, for almost every chemical or biological system, pH value is a very important factor. For some DNA structures, it is also true. Therefore, it will be very interesting to use pH change for driving DNA nanomachines.

Towards this goal, Liu and Balasubramanian (Liu and Balasubramanian 2003) invented a C base-rich DNA nanomotor (Fig. 7c) which is based on the pH-responsive folding and unfolding of a quadruple structure called i-motif. The operation of this machine, which is based on the addition of acid or base to switch solution pH values, can be completed in less than 5s and the wastes are only salt and water from neutralization reaction. The advantages of this proton-driven nanomachine are obvious: it is clear, quick, reliable and effi cient. In order to further simplify the experiment by freeing our hands from adding fuels manually, light- (Fig. 7d) (Liu et al. 2007) and electricity-controlled (Yang et al. 2010) i-motif nanomachines have also been developed. In addition, the pH-sensitive DNA triplex-duplex transitions (Chen et al. 2004), and photo-switchable G-quadruplex (Wang et al. 2010) have also been made to build DNA nanomachines.

5.2 Spatial Control of Nanoparticles by DNA Nanomachines

One important application of DNA nanomachines is to regulate the movement of nanoparticles. By controlling the change of DNA structures, the spatial arrangement of nanoparticles can be tuned accordingly (Sun et al. 2010, Chen et al. 2010). For example (Meng et al. 2009), the gold electrode is modifi ed with i-motif DNA machine which can fold into a four-stranded structure under acidic condition and unfold under basic condition. The other end of DNA is connected with CdSe/ZnS core–shell quantum dots. At acidic and basic condition, the i-motif machine can change its confi guration and brought quantum dots near/far to the gold electrode,

respectively, providing a strategy to dynamically control the photoelectric conversion. Similarly, i-motif DNA can be anchored to AuNPs to tune the assembly of AuNPs. For instance, ssDNAs containing half stretch of i-motif DNA are modifi ed onto AuNPs, and at high pH, showing a random coil structure. In contrast, at low pH, the formation of interparticle i-motifs leads to the assembly of AuNPs into aggregates. The two states can be switched by varying pH (Wang et al. 2007). Differently, when the full stretch of i-motif DNA are modifi ed onto AuNPs, there is no interparticle interaction at low pH, although the i-motif DNA folds into compact structure, and AuNPs aggregate when pH is increased (Sharma et al. 2007). Both examples show that by modifying AuNPs with DNA machines, it is possible to reversibly switch the assembly and disassembly of AuNPs. There are also other means, such as using the DNA strand displacement to control the assembly of nanoparticles and their relative positions (Song and Liang 2012, Elbaz et al.

2013). As the process is dynamic, and it allows the control of the movement of nanoparticles on a designed DNA origami pattern towards a desired direction (Gu et al. 2010).

Besides controlling spatial arrangement of inorganic nanoparticles by DNA machine, the same strategy can be applied to organic nanoparticles and more. For example, two amphiphilic dendrons are covalently connected with an i-motif DNA machine and the amphiphilic dendron contains two parts.

The yellow region represents the inner hydrophobic poly(arylether) and the blue regions represent the peripheral hydrophilic oligoethylene glycol (OEG) (Sun et al. 2010). At a basic condition, the rigid double helix formed by hybridization of DNA extend two dendrons apart for about 5.8 nm. As the pH decreases, the i-motif DNA machine folds into a compact quadruplex structure, bringing two dendrons close, so that they merge together. This process is reversible and the association of the two dendron parts increases the stability of i-motif DNA. Their increased melting temperature (T_m) is an indicator or used to probe the strength of the interaction between two nanoparticles. With the versatile modifi cation of DNA, the dendron and protein can be bound to both sides of DNA and the conformation of the dendron-DNA-protein hybrid molecular system can be controlled by the i-motif DNA machine reversibly (Chen et al. 2010). The incorporation of DNA machines into synthetic molecules or biological components provides a new platform to achieve well-defined supramolecular assemblies, manipulate nanoparticles at single molecular level in a controllable manner and study the interaction between nanoparticles.

5.3 Regulation of Protein Binding Afϔinity

Biology usually involves polyvalent interactions, i.e., the simultaneous binding of multiple ligands on one biological entity to multiple receptors

on another. Such interaction strongly relates to the spatial position of multiple ligands (Mammen et al. 1998). A DNA machine enables the study of complex interactions in three dimensions. In the early stage of this fi eld, scientists employed ssDNA and dsDNA forms to control the spatial distance (Choi and Zocchi 2006, Röglin et al. 2007, Williams et al. 2009, Furman et al. 2010). The protein entities are covalently attached to DNA strands. As ssDNA is usually considered fl exible and dsDNA is rigid with a persistence length of about 50 nm, the change of the rigidity from ssDNA to dsDNA can adjust the approximation of two proteins. It is noted that the more rigid DNA structures, such as double-crossover (DX), Holliday junction and origami scaffolds may provide more controllability and precision. For example, a tweezers-like DNA machine (Fig. 8a) can be opened (stem-loop structure) and closed (double helix structure) via adding fuel and antifuel strand, respectively (Zhou et al. 2012). Two ligands which can bind the target protein, thrombin, are introduced at both terminals of the DNA tweezers. In the closed state, the two ligands are at cooperative position and catch thrombin, however, as the DNA tweezers open, two ligands are apart and the thrombin is released. Such a target-responsive DNA machine can be repeatedly used and provides a promising strategy to study spatial dependent interactions of nanoparticles.

Figure 8. Regulation of biomolecules by DNA nanomachines. (a) Reversible regulation of target binding affi nity. Reprinted with permission from ref. (Zhou et al. 2012). Copyright 2012 American Chemical Society. (b) Regulation of enzyme cascade. Reprinted with permission from ref. (Xin et al. 2013). Copyright 2013 John Wiley & Sons Ltd.

5.4 Regulation of Enzyme Cascade Reactions

In living systems, a series of enzymatic reactions in a cascade way is conducted to mediate biological functions. The effi ciency and specifi city of enzyme cascade reactions rely on the appropriate spatial arrangement.

DNA nanotechnology provides predictable and programmable design of positioning nanoparticles in a precise manner. By taking advantage of this, previous studies have shown that it is feasible to locate multiple enzymes on static DNA nanostructures (Barrow et al. 2012). However, the distance between enzymes is determined and fi xed by the underlying DNA structures therefore, in order to mimic the enzyme cascade reaction in vitro where the location of enzymes is not consistent, scientists turned to dynamic DNA nanostructures (Xin et al. 2013, Liu et al. 2013). Following the work of using DNA tweezers to regulate protein binding, the similar design is used to attach two enzymes of glucose oxidase (GOx) and horse-radish peroxidase (HRP) to both arm ends of DNA tweezers (Fig. 8b). As the reaction occurs, GOx fi rst catalyzes oxidization of glucose to generate gluconic acid and H₂O₂, which is as the substrate of HRP and reduced into H₂O. The turnover rate of GOx is much slower than HRP, therefore, the diffusion distance of H₂O₂ determines the rate of this enzyme cascade reaction. By deliberately tuning the state of DNA tweezers in closed and open state, the distance of GOx and HRP can be adjusted from several nanometers to about eighteen nanometers. In the closed state, GOx and HRP are brought into proximity, leading to a much closer diffusion pathway for H₂O₂. In contrast, in the open state, both enzymes are spatially separated, thus lowering enzymatic effi ciency. It is shown that several cycles of regulation of enzyme cascade reaction are successfully conducted. Furthermore, this proof-of-concept is also confi rmed in another system to actuate the activity of an enzyme/

cofactor pair. A dehydrogenase and NAD⁺ cofactor are fi xed on DNA tweezers, and the enzyme inhibition and activation is reversibly turned on and off upon tuning the open and closed state of DNA tweezers, respectively. This approach opens up the design of dynamically regulating other enzyme systems and mimicking enzyme cascade reaction outside of living organisms.

Dalam dokumen and Applications of Smart and (Halaman 45-50)