NANO LETTERS
Addressable Molecular Tweezers for DNA-Templated Coupling Reactions
2006 Vol. 6, No. 5 978-983
Rahul Chhabra, Jaswinder Sharma, Yan Liu, and Hao Yan* Department of Chemistry and Biochemistry & The Biodesign Institute, Arizona State UniVersity, Tempe, Arizona 85287 Received January 27, 2006; Revised Manuscript Received March 24, 2006
ABSTRACT Here we report the construction of fully addressable DNA-based molecular tweezers to actuate coupling reactions in a programmable fashion. Three tweezers, each bearing two coupling reactants, are self-assembled on a linear DNA track. A fourth tweezer floating freely in solution can be brought to any one of the tweezers and close them by the addition of a unique pair of “fuel” DNA strands. The coupling reactions happen when the tweezers are closed, and this can be controlled sequentially from one tweezer to another. A molecular device of this kind would not only enable programmable chemical reactions but also allow distance-dependent control of biomolecular interactions.
Biomimetics and self-assembly provide effective routes for building molecular devices from the bottom-up. For example, DNA offers unique properties for assembling molecular machines because conformational changes can be easily built into sophisticated DNA nanostructures1-11 and devices,12-24 and reactant groups can be attached to DNA backbones, which have been used as a novel method for DNA-templated chemical synthesis.25 Recently, Seeman and co-worker26 have reported a design of a sequence-dependent DNA nanomechanical device that can align DNA molecules into linear polymers of different base sequences with the help of enzymatic ligation. Mao and co-workers27 also developed a strategy for switching chemical reactions on the basis of a DNA duplex-triplex transition, in response to a change of the solution pH. The above designs represent exciting steps toward building molecular assemblers to direct chemical bonding processes. Nevertheless, versatile of motion and space controls based on other conformational changes are needed to achieve more sophisticated molecular assemblers. Here we describe a new design and experimental demonstrations of a device showing sequence-dependent tweezer-like motions with addressability. The key part of the device (Figure 1) described here is a robotic arm termed as “header”, which performs a tweezerlike motion when it is triggered by a particular set of singlestranded DNA termed as the “set strand”. The tweezer can be moved from one station to another that is attached to a self-assembled track. Each station contains another tweezerlike DNA molecule (termed as “footer”) bearing a pair of coupling reactants (-NH2 and -COOH). The tweezer of a particular footer on the track will be pinched when the header binds to it and brings the two reactants adjacent to each other. * Corresponding author. E-mail:
[email protected]. 10.1021/nl060212f CCC: $33.50 Published on Web 04/07/2006
© 2006 American Chemical Society
This motion triggers a coupling reaction between -NH2 and -COOH to form an amide bond, covalently linking two DNA strands on the footer into one DNA strand. Note that although there is no chemical or physical reason for linking the three tweezers via a molecular track for the reactions we performed in this paper, we did so because it illustrates the potential for, someday, performing crosstalk-free sequential reactions on one molecule. Each DNA tweezer designed here is a four-arm branched DNA junction in which the four double helical arms of the molecule assemble into two helical stacking domains.28 It has been shown that, when free in solution containing Mg2+ cations, the four-arm DNA junction assumes a conformation with the acute angle between these two helical domains variable with a distribution of 60° ( 13° ((13° is the standard deviation of the Gaussian distribution).29 This leads to a spatial separation of the two reactants when they are each tethered at the ends of two different arms, shown in Figure 1. The spatial distance between each pair of coupling reactants is estimated to be 6.8 ( 1.4 nm when each of the two reactants is 21 nucleotides (2 full turns) away from the junction site (with 68% of chance in the range of 5.4-8.0 nm and 95% chance in the range of 4.0-9.2 nm). The distal separation between the two coupling reactants prevents the reaction from happening. One of the three different pairs of set strands (S1 and S2, S3 and S4, S5 and S6) can bring the header molecule to one of the three footers by hybridizing part of their sequences to the two single strand segments in the header molecule and another part to the two single strand segments in the footer molecules. This leads to the binding of the header molecule to the footer, and in turn they form a complex similar to a DNA double crossover motif at each station.30 Because the
Figure 1. Schematic drawing of the design. (a) A cartoon shows the nomenclature used in the design. (b) Shown in the middle is the DNA track that contains three footers, F1, F2, and F3, each linked to the track containing a linear array of DNA double crossover motifs. (It should be noted that the track can be of any desired length and number of footers; it can bear as many as allowed by the operation of the assembly.) Here the footers are separated by four helical turns, ∼13.5 nm. The red and blue dots represent the -NH2 and -COOH group covalently linked to the 3′ and 5′ ends of the two separate strands incorporated in the footers. The header (H) and footers all have singlestranded sticky ends, which each hybridize with part of the set strands (S1 and S2, S3 and S4, S5 and S6 for F1, F2, and F3, respectively). By addition of the specific pair of set strands, the header can go to any footers on the track, facilitating the formation of the amide bond coupled products (P1, P2, and P3). By addition of the pair of the unset strands (Sh 1 and Sh 2, Sh 3 and Sh 4, Sh 5 and Sh 6 for F1, F2, and F3, respectively), which removes the set strands pair from the device, the header can be released from the footers, thus ready for the next step. The assembly of header and the three footers can go in any sequence controlled by the sequence of adding the set strands or simultaneously in any combination. Note that crosstalk between tracks is possible in this design. Nano Lett., Vol. 6, No. 5, 2006
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Figure 2. (a) An 8% nondenaturing PAGE showing the formation of the complexes between the header and the footers on the track (labeled with H.F1, H.F2, and H.F3, respectively). Lane F: track with 3 footers only. Lane M: 20 bp DNA ladder. (b) AFM images showing the binding of the header on three different footers. All scale bars shown are 15 nm.
two reactants are incorporated at the nucleotides that are two full turns away from the junction site, they will become close to each other in the context of the double crossover molecule. Each of the set strands contains a short eight-nucleotide unpaired segment (“toehold”) at one end to facilitate its removal by its unset strand (represented as Sh ) that contains its fully complementary sequence through a strand displacement technique driven by branch migration.13 Figure 1 illustrates the processes involved in moving the header molecule from one footer to another. It is worth noting that the position of the header can be controlled in such a way that it can sit on any number of footers at any given time and it is also possible to move the header from one footer to another in any sequential order. We have used both nondenaturing gel electrophoresis and atomic force imaging (AFM) to characterize the device assembly. The binding of the header to the footers on the track leads to higher molecular weight complexes compared with footers on the track only and these are evidenced by the slower migration bands shown in Figure 2a. Also, the hybridization of header to the footer results in a notable topographic change that can be detected by AFM imaging, as shown in Figure 2b. To test the working principle of the device, we have performed the transition of the header molecule from footer 1 (F1) to footer 2 (F2) and then to footer 3 (F3) using the strand displacement technique. The effects of the header molecule binding on the different footers were monitored by detecting the product molecules of the corresponding coupling reaction between -NH2 and -COOH, which were 980
covalently linked at the tips of the footer molecules. The coupling reaction at each footer leads to a covalently linked longer DNA strand through an amide bond. The lengths of the coupled DNA strands are different at the three footers and are designed to be different from any of the other DNA strands on the whole device structure. Thus, the coupling reaction can be addressed easily, and the presence of the coupling reaction products can be detected by sampling the reaction mixtures onto the denaturing gel at different stages of the transition. Two experiments have been performed to test the functions of the addressable tweezer device. The difference between these two experiments is that the first experiment only produces one single product at one time, whereas the second experiment can produce multiple products sequentially. The first experiment was executed as follows: (1) Only the track with the three footers was assembled (without header). Coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium chloride (DMT-MM) was added to catalyze the amine acylation. Because there was no header and set strands present in the solution, none of the three coupling reactions can be detected due to the distal separation of the two reactants (lane F in Figure 3a); (2) The header molecule and the track assembly were mixed together in a 1:1 ratio, then the solution was divided equally in volume into three tubes. S1 and S2 were added to the solution in tube 1 to bring the header to F1. After the addition of DMTMM, the coupling reaction happened on F1, which led to a DNA fragment of ∼25 nucleotides (band labeled with red star in lane H.F1); (3) In tube 2, S1 and S2 were added to Nano Lett., Vol. 6, No. 5, 2006
Figure 3. (a) Denaturing PAGE gel showing the function of the device when the header is moved along the track and the coupling reaction was only initiated in the final step as the catalyst DMT-MM was added. Lane M: DNA marker with three known unmodified DNA single strands, the number of bases are 28, 36, and 42. Lane F: Footers without the header. No coupling reactions are observed. Lane H.F1: header on footer 1. The band at 24 comes from the set strands and the bands at 30 and 34 come from strands in the header. A new band marked by a red star shows up at ∼28 base representing the coupling product P1 (actually 25 base). Lane H.F2: header moved from F1 to F2. The new band marked by blue star shows up at ∼45 base representing P2 (actually 42 base). Lane H.F3: header moved from F1 to F2 and then to F3. The new band marked by a green star shows up at ∼42 base representing P3 (actually 38 base). (b) Denaturing PAGE gel showing the function of the device when the header is moved along the track and the coupling reaction was initiated at each step as the catalyst DMT-MM was added. Lane M: DNA Markers. F: footer alone. H.F1: header on F1. H.F2: header moved from F1 to F2. H.F3: header moved from F1 to F2 and then to F3. The product bands on F1, F2, and F3 are marked by red, blue, and green stars, respectively.
bring the header to F1 and followed by the addition of their unset strands to let the header depart from F1. S3 and S4 were subsequently added to bring the header from F1 to F2. DMT-MM was then added after this transition to trigger the coupling reaction on F2. A DNA fragment of 42 nucleotides resulting from the coupling reaction is evidenced in lane H.F2 (band labeled with a blue star). (4) In tube 3, the same process was executed to bring the header to F2, then unset strands for S3 and S4 were added to free up the header from F2. S5 and S6 were subsequently added to bring the header onto F3. After the addition of DMT-MM, the coupling reaction produced a DNA fragment of 38 nucleotides (band labeled with a green star in lane H.F3). This experiment confirmed the designed movements of the header molecule from one footer to another and revealed the functions of the device by the formation of the three different coupling products on each footer. Nano Lett., Vol. 6, No. 5, 2006
We have further performed a second experiment to test if the header molecule can trigger coupling reactions in a sequential fashion. In this experiment, the transition processes described in the first experiment were done in a single tube and samples were aliquoted for denaturing gel after the completion of each transition (i.e., after the header moves from one footer to the next footer and the subsequent addition of DMT-MM). In this way, sequentially triggered coupling reactions can be observed. In Figure 3b, lane H.F1 shows that the reaction happened on F1 when the header was bound to it. Lane H.F2 shows that reactions happened on both F1 and F2 after the header moved from F1 to F2. Lane H.F3 shows that all three coupling reactions happened when the header went through the three footers sequentially. The above two experiments clearly demonstrated the positional and sequential controls of the DNA tweezers for chemical synthesis. There are two control experiments worth 981
noting. First, when no headers were present on any footer, no reaction was detectable in the presence of DMT-MM (lane F in both gels). Second, even the header had sat on F1 or both F1 and F2 (last two lanes in Figure 3a), but before adding DMT-MM, no reaction was detected on these footers. These indicate that both the presence of the catalyst and the correct conformation of the header on footers are the essential necessary prerequisites for the coupling reaction. These also clearly verify that the tweezer-like motion of the header performed its designed function. It is noted that the product bands in the denaturing gel run slower than the pure DNA bands as compared to the unmodified DNA strands in the marker lane by ∼3 nucleotides. This can be understood that the C6 linker, which links the -NH2, and the C10 linker, which links the -COOH group to the sugar backbones of the modified DNA strands, makes the amide coupled product strands a few nanometers longer than the unmodified DNA strands of the same number of bases, and the overall charge density of the molecules are smaller than pure DNA; therefore, they migrate slower in the gel. Denaturing gel shown in Figure 3 can only give rough information on the length change of the covalently coupled DNA strands and is not accurate enough to tell the exact molecular weight of the reaction products. To further confirm that we got the correct products at all footers, we purified the reaction products from the distinct bands in the denaturing gel by gel-elution and ethanol precipitation. Then MALDI-TOF MS was used to verify the molecular weight of each product. Figure 4 shows the three MALDI-TOF MS spectra for the products corresponding to each transition. The peaks positioned at 8069, 13 382, and 11 977 for each spectrum correspond well with the expected molecular weights of the three product molecules (8071.4, 13383.8, and 11975.9), which are calculated from the initial molecular weights of the two DNA single strands containing -NH2 and -COOH groups, respectively, linked through an amide bond eliminating one water molecule. These results confirm unambiguously that the correct covalently linked products are formed from the action of the device. In summary, we have prototyped a molecular device that contains a robotic tweezer to control the production of chemical species at unique positions along a track. The performed functions on the track are positional (its location on a track is controllable), sequential (the order of the header movements can be controlled), and spatially (conformational change for both the header and footer molecule is predictable) addressable. Although not tested here, it is possible in principle to use the fuel strand displacement to remove the product out of the device and replug a new set of reaction carriers in the device without destroying the architecture of the assembly. In this design, the header can move from one track to another track once it is detached from a footer. However, it is desirable, in the future, to attach the header to the track through single-stranded hinges to prevent crosstalk between tracks. Such design could lead to a more robust molecular assembler. Although we have only chosen a simple coupling reaction to demonstrate the working 982
Figure 4. MALDI-TOF MS spectra of the purified products. (a) Product 1 from Footer 1. (b) Product 2 from Footer 2. (c) Product 3 from Footer 3. The peak with the highest intensity in each spectrum corresponds to the single charged ion of the product molecule. The other dramatic peaks with smaller m/z values are the doubly charged or triply charged ions.
principle of the device, there is no fundamental limitation to use the designed molecular assembler to control more sophisticated reactions because a rich library of DNAtemplated organic synthesis has been developed successfully.31 In addition to the use of the addressable tweezers for programmable chemical reactions, such devices may also find applications in probing cooperative interactions between pairs of tethered peptides at the tweezer-like footer by investigating cooperative effects in ligand binding. This could lead to DNA-triggered binding and releasing of molecules that will allow us to build addressable nanosensors. Acknowledgment. We thank Berea Williams for proofreading the manuscript. This work has been supported by grants from the NSF to H.Y. and a grant from Arizona State University. Supporting Information Available: DNA sequences, experimental methods, and additional AFM images. This material is available free of charge via the Internet at http:// pubs.acs.org. Nano Lett., Vol. 6, No. 5, 2006
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