Controlled Growth of DNA Structures From Repeating Units Using the

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Controlled Growth of DNA Structures From Repeating Units Using the Vernier Mechanism Andrea A. Greschner,‡ Katherine. E. Bujold,‡ and Hanadi F. Sleiman* Department of Chemistry and Centre for Self-Assembled Chemical Structures (CSACS), McGill University, 801 Sherbrooke Street West, Montréal, Canada, H3A 0B8 S Supporting Information *

ABSTRACT: In this report, we demonstrate the assembly of lengthprogrammed DNA nanostructures using a single 16 base sequence and its complement as building blocks. To achieve this, we applied the Vernier mechanism to DNA assembly, which uses a mismatch in length between two monomers to dictate the final length of the product. Specifically, this approach relies on the interaction of two DNA strands containing a different number (n, m) of complementary binding sites: these two strands will keep binding to each other until they come into register, thus generating a larger assembly whose length (n × m) is encoded by the number of binding sites in each strand. While the Vernier mechanism has been applied to other areas of supramolecular chemistry, here we present an application of its principles to DNA nanostructures. Using a single 16 base repeat and its complement, and varying the number of repeats on a given DNA strand, we show the consistent construction of duplexes up to 228 base pairs (bp) in length. Employing specific annealing protocols, strand capping, and intercalator chaperones allows us to further grow the duplex to 392 base pairs. We demonstrate that the Vernier method is not only strand-efficient, but also produces a cleaner, higher-yielding product than conventional designs.

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INTRODUCTION DNA self-assembly is a powerful method to create addressable nanostructures, with potential applications in fields such as drug delivery, diagnostics, nano-optics or molecular machines.1−4 In current approaches, complex DNA structures are generated by using a large number of unique, noninteracting DNA sequences. A particularly attractive goal would be to build well-defined DNA structures from a minimum number of identical subunits, thereby reducing the cost of this strategy and facilitating the in vivo applications of these nanostructures. Recent efforts to decrease the requirement for unique sequences have examined customized assembly protocols,5,6 insertion of synthetic organic linkers into the DNA backbone,7−13 and the use of noncovalent DNA duplex stabilizers such as intercalators.14,15 A parallel problem exists in the general field of biomacromolecule synthesis. The construction of a polymer of programmed length with a repeating number of monomers is currently challenging. Living polymerization results in length control depending on the monomer to initiator ratio,16−19 but the materials formed still possess a degree of polydispersity, and this heterogeneity increases when shorter oligomers are desired. In addition, living polymerization is not applicable to many classes of biologically relevant polymers. The Vernier mechanism is a system used to align interacting building blocks of different sizes. For example, consider two building blocks, P1 and P2. P1 has two binding sites, and P2 has three binding sites. If the two blocks bind together such that one of their ends align, the other end will not terminate at the same position. However, if three P1 units and two P2 units are aligned, © 2014 American Chemical Society

all the binding sites will be occupied (see Scheme 1). In more general terms, when the number of units in the longer building block (nP2) is not a multiple of the number of units in the shorter building block (mP1), then the shorter building block will template the growth of a larger structure with a programmed total number of units equal to n × m. The assembly of differently sized subunits to create a single, larger unit is referred to as the Vernier mechanism.20 The Vernier mechanism has been successfully applied in the field of supramolecular chemistry. For example, Bregant et al. created two molecular subunits with six hydrogen donors and four hydrogen acceptors, respectively. These assembled into the desired Vernier structure containing 24 hydrogen bonds in a 95% yield.21 The group of Hunter used zinc and tin porphyrin oligomers to produce a triple-stranded 3:4 Vernier complex.15 More recently, metal−ligand interactions were used as “units” to assemble a macrocycle which was subsequently ligated to create a structure containing 12 porphyrin units.20,22 Here we demonstrate the use of the Vernier mechanism in DNA nanoassembly23 and explore different methods of achieving clean and high-yielding products. Our units consist of a single 16-base sequence as the binding site, and repeats of this single sequence with its complement are used to assemble each strand. By varying the number of unit repeats per strand, we were able to create increasingly large final structures, whose Received: April 25, 2014 Revised: June 19, 2014 Published: June 25, 2014 3002

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Scheme 1. Vernier Assembly Mechanism

To achieve a blunt-ended structure using two different-sized interacting building blocks, a specific ratio of the two blocks is necessary. In this case, the number of binding sites in P1 is 2 while that of P2 is 3. Therefore, three P1 blocks and two P2 blocks are necessary to obtain a blunt-ended, fully bound structure.

Scheme 2. Diagram of the Three Available Binding Motifs for Thermal Denaturation

Blue represents L4, red represents S3. Black tick marks represent (4T) spacers. (A) Assembly schematic and composition of the 3:4 complex. (B) Sites as they might appear in the Vernier system. (C) Analogues created to get accurate thermal denaturation temperatures. final number of strands found in the assembled complex. For example, the final 3:4 complex, which contains a total of seven strands, was assembled using three parts of L4 and 4 parts of S3, whereas the 2:3 complex consisted of two parts L3 and three parts S2. The samples were then annealed from 95 to 4 °C using one of the annealing methods described below. Glycerine (2 μL) was added prior to loading. Gels were cast with 6% native polyacrylamide and run for 3 h (250 V, 50 mA). Thermal Denaturation Experiments. One hundred fifty microliters of each sample was prepared for thermal denaturation (TM) analysis, with a final ssDNA concentration of 5 μM. Samples were prepared such that the TM’s of a single 16 base unit, two consecutive 16 base units, and three consecutive 16 base units could be individually determined (see Scheme 2C, SI). To prevent evaporation at high temperatures, approximately 50 μL of silicone oil were gently added to the top of each sample. The Peltier temperature controller was set to ramp the temperature at 0.5 °C/min, from 20 to 95 °C, then back down to 20 °C. Data was gathered every 0.1 °C.

length is programmed by the number of repeats in the building blocks. Employing tailored annealing techniques and intercalators, we show further increase of the yields of the Vernier products. Finally, by stabilizing the two blunt ends of the Vernier product, a 384 base pair (bp) assembly could be consistently created from the 16-base sequence. The Vernier mechanism, combined with the yield-enhancing techniques presented here, introduces a new approach to increasing the functional complexity of DNA nanostructures without relying on sequence uniqueness.

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MATERIALS AND METHODS

System Design. To validate the potential of the Vernier mechanism for DNA nanoassembly, the simplest possible structure was chosen: a linear duplex. The linear system consisted of two building blocks, each with repeats of a single sequence making up the “units” of each block. Each unit contained 16 bases, chosen for minimal intrastrand interactions, and was separated from the next unit by four thymidine spacers. In each system, the longer of the building blocks was designated the long strand (LX, where X is the number of units in the strand). The shorter building block was designated SY, where Y is the number of repeat units, each of which is complementary to the units in LX. Polyacrylamide Gel Electrophoresis (PAGE) Analysis. Samples for native gel analysis were prepared with total single-stranded DNA content of 0.1−0.2 nmol (SybrSafe staining) or 0.0025−0.01 nmol (GelRed staining) to a total volume of 10 μL in tris-acetic acid buffer (1xTAMg, 40 mM Tris(hydroxymethyl)aminomethane, 7.6 mM magnesium chloride hexahydrate, 1.4 mM glacial acetic acid, pH 8). In order to assemble a chosen complex, the two strands composing it were mixed in appropriate ratios. These ratios were determined from the

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RESULTS AND DISCUSSION Simple Assembly. The first assembly consisted of a long strand three units in length (L3) and a shorter block that was two units long (S2). A Vernier assembly of these two strands should contain 2xL3 and 3xS2, which we refer to as a 2:3 complex. If properly assembled, the final complex should be 112 bp long (Figure 1). Initial assembly was performed in tris-acetic acid buffer with a total single-stranded DNA content (ssDNA) of 0.2 nmol and a total volume of 10 μL. The proper ratio of 2xL3 per 3xS2 was used, and the sample was annealed (95 to 4 °C, 3.5 h, Anneal protocol 1, see SI). Product distribution was determined using nondenaturing PAGE.

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Figure 1. Assembly of 2:3 complex alone, and in the presence of ethidium bromide. (A) Assembly schematic and composition of the 2:3 complex. (B) Nondenaturing PAGE experiment demonstrating the increase in the desired 2:3 Vernier product upon incubation with EtBr. Blue lines represent the L3 strands and red represents the S3. Black lines represent 4xthymidine (4T) spacers. Lane (1) 2xL3 + 3xS2 and (2) 2xL3 + 3xS2 annealed with ethidium bromide. (C) Analyses of band intensities and yield as performed using ImageLab software. The lane in question is shown horizontally below each graph of intensity. (Inset) The structure of ethidium bromide.

Figure 2. Melting temperatures for building blocks with varying numbers of binding sites. Thermal denaturation temperatures for one binding site (yellow), two binding sites (blue), and three binding sites (red).

The 2:3 complex assembled in 44% yield, with the remainder of the DNA forming 2:2 complex or 1:1 complex. Bands were identified using sequential assembly of L3 and S2, as described in the SI. Considering the multitude of possible products (approximately eight different variations were possible, in addition to numerous oligomeric and/or polymeric products),

44% is a significant yield (Figure 1). However, to make the Vernier mechanism feasible for widespread use, a higher yield was desired. Ethidium Bromide as a Guide. Previous work by the authors15 demonstrated that addition of an intercalator to DNA systems during assembly favors the formation of fully bound 3004

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analogue. Again, each of the four anneal protocols were tested to determine which was best suited to the conventional design. Anneal Protocol 4 (an isothermal anneal using denaturants) gave slightly better results than the thermal anneals and was used for further comparisons (SI, Figure 3).

duplex structures. Since the Vernier product is the one in which the most bases are paired, it should be favored by EtBr addition. Two microliters of 10 mM EtBr solution in 1xTAMg were added to L3, followed by the addition of S2 to a total volume of 12 μL, which was annealed as before. Native PAGE analysis reveals that the yield of the desired Vernier product increases to 75% with the use of an EtBr guide. We previously demonstrated a method to remove the ethidium bromide from DNA assemblies while keeping the integrity of these structures.15 Larger Vernier System. With the 2:3 complex assembling with good yield, we moved on to a larger Vernier system consisting of three L4 and four S3 strands, creating a 3:4 Vernier complex with a total of 228 bases (Scheme 2). Simple annealing resulted in a yield of 28%, with a variety of lower-order products forming. Annealing with EtBr with this system did not result in a significant increase in yield, thus further solutions were sought. Improving Yield with Annealing Protocols. As in the previous system, a slow annealing protocol from 95 to 4 °C over 3.5 h was initially used for assembly (Anneal protocol 1). However, recent studies have indicated that a slow, steady anneal is not necessarily the highest yielding.5,6 Sobczak et al. discovered that large origami structures can be efficiently assembled by incubating the constituent strands at a constant temperature corresponding to the lower temperature boundary of the folding peak. To estimate our assembly temperature boundaries, we used thermal denaturation experiments for three different duplexes: (i) an analogue representing one 16 base pair (bp) binding site with overhangs of unique sequence on one side, (ii) an analogue representing two contiguous binding sites (with the requisite four thymidine spacers), and (iii) an analogue with three contiguous binding sites (again, with the requisite thymidine spacers; see Scheme 2). Results are given in Figure 2. It is interesting to note the increase in melting temperature as the number of binding sites increases, even though each binding site is separated by four, nonhybridized thymine spacers. This is consistent with our previous studies on cooperative melting.7 Here, we observe that a single binding site melts at 64 °C. Having two binding sites connected by a four-thymidine spacer on each strand melts at 71 °C, whereas three binding sites similarly connected will yield a TM of 74 °C. With these results, several new annealing protocols were developed. The most successful incorporated a slow temperature decrease over the TM range between 85 and 60 °C (Anneal protocol 2: 5 min at 95 °C, 85−60 °C at 3 min/°C, 59−10 °C at 1 min/°C). This protocol resulted in a yield of 32 ± 1%. While the yield was not significantly improved from our first anneal protocol, it was more rapid (a total of 2 h instead of 3.5 h for Protocol 1). As such, all future experiments were conducted using either annealing protocol 1 or 2 (see section 6 in SI for significant yield optimization procedures and corresponding PAGE experiments). Comparison of the Vernier Approach with a Unique Sequence System. With optimized assembly conditions, we were interested in comparing the Vernier approach of DNA assembly to conventional methods, which would use unique sequences. For comparison, we designed an analogous 3:4 complex in which each 16 base region had a different and unique sequence. This necessitated the identification of 12 16-base sequences (and their complements) that were not prone to missassembly (see Supporting Information). These sequences were assembled into seven strands, of which one of each was required to create the 3:4

Figure 3. Comparison between conventional assembly methods and the Vernier mechanism for the assembly of higher order structures. (Left) Conventional methods of DNA design would require the synthesis of seven different strands with 12 noninteracting sequences. Because of their complexity, several side-products are still formed. The overall yield of the fully assembled structure is low. (Right) Vernier-type assembly results in higher yields, requiring only two unique strands and one sequence.

When compared to the conventional system, not only is the Vernier approach better-yielding (with more than twice the yield), but it is also much simpler to design, with only one sequence necessary (compared to 12 otherwise). In addition, only two unique strands are required to construct the Vernier system, whereas it would be necessary to synthesize or purchase seven strands using the traditional DNA analogue. Capped Vernier Assembly for Improved Yield. Previous studies24 have suggested that use of a blunt-ended nucleation site might enhance the yield of a Vernier complex. This rationale can be explained through Scheme 3. There are several ways in which an L4 and S3 strand can initially bind. Some pairings have three binding regions (A and D), some have two (B and C). To construct a 3:4 complex, two blunt-ended, three-region dimers (A,D), and only one two-region dimer (B,C) are required. By preassembling the proper “starting” building blocks (A or D in our case), a nucleating scaffold from which a complete Vernier structure can assemble is provided. In an effort to improve this strategy, a capping method was devised. This method consists of breaking the assembly mechanism into two steps. First, the edges of the construct are annealed, followed by the assembly of the middle portion. The goal was to provide preassembled building blocks with the correct offset for the other units to come together and yield a blunt-ended construct. We thus designed capping strands with two 24mer binding regions (C4-1 and C4-2) instead of the three 16 base stretches that were used previously. During the anneal, these 24mer segments would come together with their complements first (C4′) and then direct the assembly of the 3005

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Scheme 3. Required Units for the Assembly of a 3:4 Vernier Complex

(Top) A 3:4 Vernier complex. (Box) A variety of possible L4 and S3 pairings. A and D are blunt-ended, three-region pairings. One of each is required for assembly of a 3:4 complex. C and D are two-region pairings. Only one or the other is required for assembly (combined with an extra S3 strand).

Scheme 4. Schematic of a Capped Vernier 3:4 Complex

(A) The capping units have a higher annealing temperature than the traditional Vernier building blocks, allowing them to assemble first during the heat/cool cycle. (B) Full assembly schematic of the capped 3:4 complex. (C) Comparison of product distribution for the uncapped L4 + S3 system and the capped system. The capped system assembles more cleanly.

We saw an improvement in yield as well as a reduction in the product distribution when the capping method was applied. Briefly, three new strands (C5−1, C5−2 and C5′, Scheme 5) were designed to replace the edges of the complex and added to the other building blocks so that the ratios between each strand were equivalent. C5−1 and C5−2 are composed of three 24 base pair regions to replace the four 16mer stretches present on the initial strands, and are terminated with a 16mer region at the 3′ or the 5′-end (respectively) to make them compatible with the repeating units in the system. The third strand, C5′, is complementary to their 24-base binding regions. Using the capping method in combination with the addition of EtBr prior to the anneal, the product distribution was narrowed, and the yield of the desired structure increased to 50 ± 10% (compared to an uncapped Vernier annealed with EtBr at 11 ± 7%, Scheme 5). Note that this method uses only two repeat sequences. For comparison, synthesizing a single comparable nucleotide of full length would, assuming 99.5% coupling efficiency, provide a final synthesis yield of only 14%. Current automated synthetic techniques are unable to reliably produce strands of that length.

repeating 16-base Vernier segments in the middle (Scheme 4). The result is a system with blunt-capped ends and a Vernierassembled center. Assembly of the capped system resulted in a larger proportion of the desired product and fewer side products, as compared to the conventional L4 + S3 system. To test the scalability of the Capped Vernier approach, we attempted to assemble a larger structure, the 4:5 complex. When assembled, this complex will be composed of four long strands, each containing five binding units (S5, five binding sites) and five tetramers (L4, four binding sites) for a total of 384 bases. A first attempt at assembling this structure was done by mixing components S5 and L4 in a 4:5 ratio, respectively, and without capping strands. The component strands were mixed and subjected to a 3.5-h thermal anneal from 95 to 4 °C (anneal protocol 1) in the presence of EtBr. This method afforded the expected product in 11 ± 7% yield along with a range of partially assembled structures. The 4:5 complex was identified through comparison with previous Vernier systems (primarily the 3:4 complex) and use of a DNA ladder. 3006

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Scheme 5. Comparison between the Uncapped and Capped 4:5 Vernier Complexes

(A) Assembly of S5 and L4 into the uncapped 4:5 vernier complex. Underneath, the assembly of the capped 4:5 Vernier complex is shown. Strands are identified by color. (B) Native PAGE assembly of both the uncapped and capped 4:5 Vernier systems. Addition of EtBr both narrows the product distribution and increases yield, especially in the capped system.

intercalator, lengths of up to 392 bp (Capped 4:5) were obtained. This work will establish a basis for future DNA nanostructure designs that rely less on sequence design, allowing for more focus on functionality. More generally, the Vernier concept can be applied to other classes of supramolecular polymers,25 in which alignment of two interacting units of different sizes may allow control of chain length and molecular weight distribution.

The longest strand used in the 4:5 capped Vernier is 100 bases, which has a synthetic yield of ∼60%. For this optimized Vernier system, the assembly yield is 50%, which equates to a 30% product yield from initial reagents. This is twice as much product as could be attained from a single nucleotide−even if the assembly is perfectly clean. These results suggest that providing preassembled and properly offset building blocks favors the full assembly of larger order structures. In the case of the 4:5 assembly, we can create a duplex 392 bp long using only 2 DNA sequences (one sequence for the 16mer repeats in the S5 strands and one 24mer sequence for the capped strands). This significantly decreases the amount of strands and sequence design usually required for a system of this length.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental methods, DNA sequences, assembly protocols, and materials. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSION We have demonstrated the use of the Vernier mechanism toward DNA nanostructure assemblies to produce larger order structures with programmed lengths. Using only a 16-base building block and its complement and varying the number of repeats per strand, we created Vernier constructs ranging from 112 bp (2:3 complex) to 228 bp (3:4 complex). Comparison with conventional methods of sequence-design based on the generation of unique sequences shows that the Vernier approach is not only simpler, but higher-yielding. By optimizing annealing conditions, adding capping strands, and guiding assembly with an

AUTHOR INFORMATION

Corresponding Author

*Fax: +1-514-398-3797; Tel: +1-514-398-2633; E-mail: hanadi. [email protected] Author Contributions ‡

These authors contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 3007

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ACKNOWLEDGMENTS We would like to thank NSERC, FQRNT, CSACS, CFI and CIHR.

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REFERENCES

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