Communication Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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DNA-Functionalized, Bivalent Proteins Janet R. McMillan and Chad A. Mirkin* Department of Chemistry and International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States S Supporting Information *
dictated by particle shape.9,10 However, the synthesis of DNAfunctionalized building blocks where valency, defined as the number of particle binding events in which a building block can engage, and the directionality of this binding can be controlled separately from shape, and realized with small numbers of oligonucleotides, is under-explored. Proteins, however, offer an ideal platform to realize directional bonding and valency control in nanoscale building blocks, as they offer the ability to engineer constructs with nearly arbitrary patterns of oligonucleotides because of their tunable surface chemistry. At the same time, proteins represent important synthons for preparing functional supramolecular materials. We11−13 and others14,15 have explored the assembly of isotropically, or highly functionalized proteins; however, realizing directional interactions with small numbers of oligonucleotides strategically placed on the periphery of a protein building block remains unexplored, but could represent a powerful, and general strategy to realize nanoscale building blocks with programmed valency. Here, we report the design and synthesis of a DNAfunctionalized protein with valency controlled through the placement of DNA bonding elements at opposing ends of the structure. We demonstrate that only two surface-conjugated oligonucleotides are sufficient to achieve directional DNA bonding, which is important for generalizing this assembly approach to proteins with small edge lengths that cannot accommodate large numbers of DNA modifications. We introduce a new platform to synthesize nanoscale building blocks where both valency and directionality can be defined in a manner entirely controlled through exploiting the addressable and tunable chemical topologies of protein surfaces. Synthesizing DNA−protein building blocks that display directional bonding and defined valency with few oligonucleotide modifications requires careful design considerations: whereas anisotropic nanoparticles offer large surface areas that enable the cooperative hybridization of hundreds of oligonucleotides and therefore a significant thermodynamic driving force toward directional particle assembly that maximizes the number of hybridization events,9 protein faces have surface areas reduced by almost an order of magnitude and can only accommodate a handful of DNA modifications, thus reducing the driving force for directional assembly. To address this challenge, we turned to the array of literature investigating the design parameters that favor the cooperative hybridization of small numbers (2−3) of oligonucleotides appended to smallmolecule organic cores.16−18 From this, we hypothesized that positioning a pair of short oligonucleotides (10 base pairs) less
ABSTRACT: Bivalent DNA conjugates of β-galactosidase (βGal), having pairs of oligonucleotides positioned closely on opposing faces of the protein, have been synthesized and characterized. These structures, due to their directional bonding characteristics, allow for the programmable access of one-dimensional protein materials. When conjugates functionalized with complementary oligonucleotides are combined under conditions that support DNA hybridization, periodic wire-type superstructures consisting of aligned proteins form. These structures have been characterized by gel electrophoresis, cryotransmission electron microscopy, and negative-stain transmission electron microscopy. Significantly, melting experiments of complementary building blocks display narrowed and elevated melting transitions compared to the free duplex DNA, further supporting the formation of the designed binding mode, and unambiguously characterizing their association as DNA-mediated. These novel structures illustrate, for the first time, that directional DNA bonding can be realized with only a pair of DNA modifications, which will allow one to engineer directional interactions and realize new classes of superstructures not possible simply through shape control or isotropically functionalized materials.
T
he synthesis of nanoscale building blocks with precisely defined, directional interactions is a significant chemical challenge. In biological systems, directional, non-covalent interactions between proteins direct their assembly into functional higher order architectures, ranging from actin filaments1 to bacterial S-layers,2 and are responsible for a host of dynamic functions. The complexity realized through the well-defined interactions displayed by these biological building blocks has inspired efforts to synthesize nanoscale building blocks that display similarly well-defined directional bonding characteristics or valency.3 However, the supramolecular interactions that govern directional association of proteins in nature are highly chemically complex, and difficult to harness synthetically.4 In contrast to the complex directional interactions at play between proteins in biological systems, DNA assembles in a highly specific and predictable manner, and has proven to be a powerful tool to program interactions between nanoscale building blocks.5−8 In this regard, anisotropic nanoparticles densely functionalized with DNA can display directional bonding characteristics, where the cooperative hybridization of hundreds of strands on particle faces results in valency © XXXX American Chemical Society
Received: March 28, 2018
A
DOI: 10.1021/jacs.8b03403 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society
matrix-assisted laser desorption−ionization mass spectrometry (MALDI-MS) (Figure 1). The circular dichroism spectrum of
than 4 nm apart on a protein surface would be sufficiently close to provide a significant driving force for their cooperative hybridization, and therefore the face-to-face interaction of the proteins over branched binding modes.19,20 To test this hypothesis, we sought to design a bivalent DNA−protein conjugate that displays directional DNA bonding to assemble into one-dimensional (1D) segmented wire-like materials, as these protein structures have important implications in catalysis, sensing, and as components of artificial extracellular matrices.21 As a model protein, we selected the D2 symmetric, tetrameric protein β-galactosidase (βGal) which we have previously shown to be robust to DNA functionalization.12,22 To engineer a protein that, upon DNA modification, displays the desired bonding characteristics, ideal DNA modifications sites were identified to be close to a subunit interface of the protein, which by virtue of the protein’s inherent symmetry would result in a conjugate with two closely positioned oligonucleotides on opposing faces. We selected residue T265 as an optimal position for DNA modification, which would result in a conjugate with two DNA modifications on the top and bottom face of the protein positioned approximately 3.7 nm apart (Scheme 1). To achieve selective DNA labeling at this
Figure 1. Characterization of βGal1D-DNA conjugates. (A) Denaturing gel electrophoresis of unmodified βGal1D (1), βGal1DDNA A (2), and βGal1D-DNA B (3). (B) MALDI-MS spectra of unmodified βGal1D (green) and βGal1D-DNA B (blue).
Scheme 1. (A) Side (Left) and Top (Right) View of βgal1D, Showing Designed C265 Mutation in Yellow, and (B) DNA Modification of βgal1D with Maleimide-Terminated DNA, Resulting in the Selective Functionalization of C265
the mutant protein exhibited no observable deviations from that of the native protein, indicating that the secondary structure of the mutated protein remains intact (Figure S1). Furthermore, the size-exclusion chromatogram of βgal1D and negative-stain TEM analysis support the retention of the native, tetrameric structure of the protein in our designed mutant (Figures S2 and S3). To prepare the desired protein−DNA conjugates, βGal1D-DNA A and βGal1D-DNA B, an excess of two complementary maleimide-terminated 10-base-pair oligonucleotides (DNA A, DNA B) were separately reacted with the protein (SI 2.2, Table S2). Upon purification of the conjugate from free DNA via size exclusion chromatography (SEC, Figure S4), SDS-PAGE analysis of the protein revealed a discrete band with a slight decrease in electrophoretic mobility, which we confirmed corresponded to the addition of a single DNA strand to the monomer of the protein via MALDI-MS (Figure 1B), as designed. UV−vis extinction spectra of the protein−DNA conjugates showed an extinction maximum at 275 nm, compared with 280 nm for the unmodified protein, that is consistent with the expected number of surface-conjugated oligonucleotides (Figure S5, Table S3). Furthermore, agarose gel electrophoresis of the unconjugated and DNA-modified protein under native conditions revealed an increase in electrophoretic mobility upon DNA conjugation, consistent with the increased negative surface charge resulting from DNA conjugation (Figure S6), and analytical SEC analysis demonstrated a decrease in retention volume for the conjugates (Figure S7). Taken together, these data unambiguously demonstrate the successful conjugation of the desired number of oligonucleotides to the designed protein. Having synthesized and characterized the desired conjugates, we then investigated their association behavior. First, we combined complementary DNA-functionalized proteins in an equimolar ratio, and analyzed the resulting sample via native agarose gel electrophoresis. Here, we observed that this sample ran as a diffuse band suggestive of high molecular weight aggregates, whereas the individual βGal1D-DNA samples ran as discrete bands with faster electrophoretic mobility (Figure S9), confirming that the building blocks aggregate only in the presence of complementary DNA interactions.
site, we designed a mutant of βGal, βGal1D, wherein we replaced all solvent accessible cysteine residues with serine residues, and mutated the native T265 residue to a cysteine, providing a selective chemical handle for conjugation at the desired position (Scheme 1, Table S1). βGal1D was expressed in a bacterial expression system and purified using a combination of nickel affinity and size-exclusion chromatography (Supporting Information (SI) 1.2). The purity of the isolated protein was confirmed using SDS-PAGE and B
DOI: 10.1021/jacs.8b03403 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society
between proteins are possible. Therefore, upon combination of complementary building blocks, which formed disordered aggregates at room temperature (Figure S11), we subjected the system to a slow-cool annealing protocol to allow the system to reorganize to its thermodynamically favored state. Remarkably, cryo-TEM imaging of samples subjected to this slow-cool annealing protocol revealed the formation of the expected 1D assemblies, confirming our hypothesis that 3.7 nm spacing between pairs of oligonucleotides would result in the directional assembly of the protein (Figure 3B). Importantly,
To characterize the temperature dependence of this association, and lend insight into the binding behavior of the designed DNA−protein conjugates, we conducted melting experiments of complementary βGal1D-DNA building blocks using a donor-quenching FRET-based technique.23 Here, βGal1D was functionalized with a cyanine-3- and -5-modified DNA A and DNA B, respectively (Figure S8), and the FRET efficiency of the system, and thereby degree of association, was measured as a function of temperature (see SI 4.3 for details). Because of the close proximity of the DNA strands on each protein face, we hypothesized that the desired face-to-face binding mode of these designed building blocks would result in a significantly elevated and narrowed melting transition compared to the free oligonucleotide sequence.16 The temperature dependent association profile of complementary βGal1DDNA building blocks displayed a main transition with a Tm of 41.5 °C and full-width at half-maximum (fwhm) of 8.1 °C, compared to a Tm of 31.7 °C and a fwhm of 11.8 °C for the free DNA duplex (Figure 2). This result suggests that the designed
Figure 2. Temperature-dependent association of βGal1D-DNA building blocks. (A) Fraction assembled vs Temperature. (B) First derivative of assembled fraction vs temperature curves. Bottom right shows legend for plots: complementary (green) and non-complementary βGal1D-DNA building blocks (gray), and free DNA (black).
Figure 3. TEM characterization of βGal1D-DNA assemblies. (A) Assembly schematic, (B) cryo-TEM image of 1D assemblies, and (C,D) negative stain TEM images of 1D assemblies. Scale bars = 500 nm for (B) and (C), and 200 nm for (D). Insets show cartoon of protein along its 17 × 8 nm face, scaled to image.
building blocks interact in a face-to-face manner, with oligonucleotides on each face of the protein hybridizing cooperatively (see SI 4.3 for additional discussion). To confirm that this temperature-dependent FRET signal obtained for complementary βGal1D-DNA building blocks was a result of DNA-hybridization, and not nonspecific protein interactions or aggregation, we conducted the analogous FRET experiment wherein we combined proteins functionalized with a Cy3- and Cy5-modified single (non-complementary) oligonucleotide sequence. This sample displayed no FRET signal over all measured temperatures, confirming that the observed assembly of DNA-functionalized βGal1D is strictly DNA-mediated (Figure 2). We next investigated the structure of these assemblies via transmission electron microscopy (TEM), testing our hypothesis that the designed DNA functionalization pattern would favor the directional, bivalent assembly of our protein. In the designed system, we hypothesized that the desired face-to-face binding should be thermodynamically favored and therefore be adopted if the system is given sufficient time and thermal energy to reorganize, even though multiple binding modes
cryo-TEM micrographs of individual βGal1D-DNA samples showed the presence of well-dispersed proteins, in agreement with gel electrophoresis results (Figure S12). We also investigated the structure of these assemblies via negative stain TEM, where we again observed the formation of 1D assemblies (Figure 3C,D, Figure S13). Analysis of the length distributions of the assemblies gave weight-averaged degree-ofassembly lengths that were in close agreement with both imaging techniques (5.60 and 5.89 for negative stain and cryoTEM respectively; Figure S14). Significantly, both cryo-TEM and negative stain TEM micrographs confirm that the orientation of the protein with respect to the direction of assembly is consistent with our system design, and unambiguously demonstrate that we have programmed directional interactions between proteins with only two oligonucleotide modifications on each protein face (Figure 3B,D insets). Herein, we have synthesized and characterized bivalent DNA-functionalized protein building blocks, where we demonstrate that with careful selection of only a single amino acid mutation site, we can program their directional assembly C
DOI: 10.1021/jacs.8b03403 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society
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into 1D materials with defined protein orientation. This approach, because it requires only a pair of oligonucleotides to achieve directional assembly, has the potential to be a highly generalizable method, compared to other recent and impressive examples that require significantly higher numbers of oligonucleotides.14 Importantly, the approach introduced herein offers many advantages over other supramolecular strategies to assemble 1D protein materials, such as the use of metal-coordination,24 protein−ligand,25,26 or host−guest interactions:27 the length, binding strength, and flexibility of DNA bonds can be modulated, which we will explore in future work. Additionally, this approach presents the possibility of engineering multiple orthogonal interaction pairs on protein surfaces to access higher-order, hierarchically assembled materials. Other approaches that focus on using DNA tile and origami templates to organize proteins into extended lower dimensional materials typically do not involve well-defined protein conjugates or well-defined protein orientation.28−33 Most significantly, our results demonstrate the power of harnessing the mutability of protein surfaces to engineer nanoscale building blocks with specific valency realized through directional interactions. This approach will enable the systematic exploration of the emergent properties of DNA-functionalized nanoparticles, most notably cooperativity, and provide a platform to assemble complex structures from a diverse array of nanoscale building blocks.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b03403. Methods, Tables S1−S5, and Figures S1−S14 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Chad A. Mirkin: 0000-0002-6634-7627 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge support from the Vannevar Bush Faculty Fellowship program sponsored by the Basic Research Office of the Assistant Secretary of Defense for Research and Engineering and funded by the Office of Naval Research through grant N00014-15-1-0043. This work made use of the EPIC, Keck-II, and/or SPID facilities of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205), the MRSEC program (NSF DMR-1121262) at the Materials Research Center, the Keck Foundation, and the State of Illinois, through the International Institute for Nanotechnology. J.R.M. gratefully acknowledges the National Science and Engineering Research Council of Canada for a postgraduate fellowship.
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REFERENCES
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DOI: 10.1021/jacs.8b03403 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
