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Heterochiral DNA Strand-Displacement Circuits Adam M. Kabza, Brian E. Young, and Jonathan T Sczepanski J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10038 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017
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Heterochiral DNA Strand-Displacement Circuits Adam M. Kabza‡, Brian E. Young‡, and Jonathan T. Sczepanski* Department of Chemistry, Texas A&M University, College Station, TX, USA
Supporting Information Placeholder ABSTRACT: The absence of a straightforward strategy
to interface native D-DNA with its enantiomer L-DNA — oligonucleotides of opposite chirality are incapable of forming contiguous Watson–Crick (WC) base pairs with each other — has enforced a “homochiral” paradigm over the field of dynamic DNA nanotechnology. As a result, chirality, a key intrinsic property of nucleic acids, is often overlooked as a design element for engineering DNA-based devices, potentially limiting the types of behaviors that can be achieved using these systems. Here, we introduce a toehold-mediated stranddisplacement methodology for transferring information between orthogonal DNA enantiomers via an achiral intermediary, opening the door for “heterochiral” DNA nanotechnology having fully-interfaced D-DNA and LDNA components. Using this approach, we demonstrate several heterochiral DNA circuits having novel capabilities, including autonomous chiral inversion of DNA sequence information and chirality-based computing. In addition, we show that heterochiral circuits can directly interface endogenous RNAs (e.g. microRNAs) with bioorthogonal L-DNA, suggesting applications in bioengineering and nanomedicine. Overall, this work establishes chirality as a design parameter for engineering dynamic DNA nanotechnology, thereby expanding the types of architectures and behaviors that can be realized using DNA.
Dynamic DNA nanodevices1 and circuitry2 almost invariably represent homochiral systems comprised exclusively of D-DNA, the naturally occurring stereoisomer (Figure 1a).3 This is despite inferred similarities between D- and L-DNA in terms of solubility, hybridization kinetics, and duplex thermal stability.4 The key challenge associated with integrating both enantiomers of DNA into a single device is their inability to form WC base pairs with each other.5 While this property alone can be beneficial, especially for applications related to biotechnology4c,6, it precludes the sequence-specific transfer of information between the two enantiomers of DNA, thus undermining the interfaced (or heterochiral) construc-
tion sought here. We reasoned that this limitation could be overcome by employing an achiral nucleic acid analog as a sequence-specific mediator between the two orthogonal enantiomers of DNA. Therefore, we turned our attention to peptide nucleic acid (PNA) (Figure 1a).7
Figure 1. Heterochiral DNA strand-displacement reactions. (a) The three types of nucleic acids used in this work. DDNA (black), L-DNA (blue), and PNA (green) are distinguished by color throughout the text. (b, c) Mechanisms for heterochiral strand-displacement reactions A (b) and B (c). DNA and PNA strands (in all figures) are depicted as lines with the half arrow denoting the 3′ end (or C-terminus for PNA), and an asterisk indicating complementarity.
PNA is an oligonucleotide analog in which the sugarphosphate backbone has been replaced with uncharged N-(2-aminoethyl)glycine units. Like DNA and RNA, PNA obeys WC base pairing rules, forming stable antiparallel duplexes with DNA.8 In contrast to the native polymers, however, PNA has no inherent chirality, and as a result, hybridizes to DNA or RNA irrespective of chirality.5a,9 On the basis of this property, we conceived two novel toehold-mediated strand-displacement reac-
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Figure 2. A heterochiral DNA strand-displacement circuit implemented with Reaction A. (a) Schematic illustration of the heterochiral circuit. (b) Fluorescent reporter strategy. Domain 3* of the output strand from (a) (D-OUT1 or L-OUT1) will react with toehold domain 3 of the corresponding reporter complex (D-R1 or L-R1, respectively), displacing the incumbent fluorescent strand. (c) Potential reaction pathways (P1–P6) for the circuit depicted in (a). (d) Demonstration of the heterochiral circuit (pathways P2, P3, and P5). Unless stated otherwise, all reaction cascades contained 500 nM of the indicated circuit components, 300 mM NaCl, 1 mM EDTA, and 10 mM Tris (pH 7.6) and were carried out at 23 °C. All reaction mixture also contained 500 nM of each reporter complex (D-R1 and L-R1) and their corresponding fluorescent signals were monitored in parallel. Fluorescence (Fluor.) in all figures is reported in units such that 1.0 is the fluorescence of the triggered reporter (in this case 500 nM) and 0.0 is the background of the quenched reporter complex. Sequences of strands are listed in Table S1.
tions (hereinafter referred to as simply stranddisplacement reactions) that exploit DNA/PNA heteroduplexes in order to interface the two enantiomers of DNA (Figures 1b and 1c). In Reaction A, a DNA/PNA substrate complex (S1) is activated via binding of a DNA input (IN0) to its DNA toehold domain (t*), resulting in displacement of the achiral PNA strand. In this reaction, the chirality of input signal must match the chirality of the incumbent DNA strand on the DNA/PNA heteroduplex. Release of the PNA strand effectively decouples the stereochemical information from the sequence information present in the chiral input. At this point, the achiral PNA output can serve as a sequence-specific input for downstream reactions with either D-DNA or LDNA components. In Reaction B (Figure 1c), the toehold domain (t*) resides on the achiral PNA strand in the initial DNA/PNA substrate complex (S2). Therefore, S2 can be activated by either D-DNA or L-DNA inputs (DIN0 or L-IN0, respectively) to directly generate an output having a single, predetermined chirality. Using Reaction A as a starting point for developing a heterochiral DNA nanodevice, we designed a multilayer DNA strand-displacement circuit employing both enantiomers of each reaction component (Figure 2a). The
input strand (e.g. D-IN1) reacts with the chimeric DNA/PNA complex (e.g. D-A1) via toehold domain (1), resulting in displacement of the achiral PNA intermediate (PNA1). The activated domain (2*) on the PNA serves as the input for a second reaction with complex B1 (e.g. D-B1), releasing the output strand (e.g. D-OUT1). Depending on the chirality of the DNA components provided, the circuit can proceed through several possible reaction pathways (Figure 2c). For example, a circuit comprised solely of components D-A1 and D-B1 can only generate D-OUT1 in the presence of D-IN1 (pathway P1). This is analogous to the traditional, homochiral approach (i.e. if the achiral PNA were to be replaced with a DDNA strand having the same sequence). However, addition of L-B1 to the same reaction mixture enables simultaneous production of both enantiomers of OUT1 (pathway P3, Figure 2c), each of which can carry out unique downstream functions. To confirm our design, we first examined pathways P2 and P5 in isolation (Figure 2c), both of which produce a DNA output having the opposite chirality as the input. In order to stereospecifically monitor the outputs of the circuit, we utilized a pair of chiral reporter complexes (D-R1 and L-R1) having unique fluorophore-quencher
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pairs (Cy3/BHQ2 and Cy5/BHQ3, respectively) (Figure 2b). As shown in Figure 2d, when D-IN1 was added to a reaction mixture containing only pathway P2 components (D-A1 and L-B1), along with both reporter complexes, only the fluorescent signal corresponding to activation of L-R1 (Cy5) was observed. Likewise, only reporter D-R1 (Cy3) was activated when L-IN1 was added to a reaction mixture containing solely pathway P5 components (L-A1 and D-B1). In contrast, no significant fluorescent signal was observed for either reaction in the absence of an input or in the presence of an input with the opposite (orthogonal) chirality relative to complex A1. These fluorescent data were further verified by gel electrophoresis (Figure S1). Together, these results confirm that pathways P2 and P5 of the heterochiral circuit function as intended, and demonstrate for the first time an autonomous heterochiral DNA device. Having demonstrated the proper function of pathways P2 and P5 in isolation, we combined their corresponding components into a single reaction mixture in order to construct the complete heterochiral circuit depicted in Figure 2a. All reaction components (D-A1, L-A1, D-B1, and L-B1), as well as both reporter complexes (D-R1 and L-R1), were present at equimolar concentrations (500 nM). Addition of one equivalent of D-IN1 (500 nM) to the racemic circuit resulted in generation of approximately half the maximal fluorescent signal for each reporter complex (Figure 2d, pathway P3), suggesting that ~0.5 equivalent (~250 nM) of each output strand was produced relative to the input. This observation is consistent with the equal consumption of the achiral PNA strand (PNA1) by each enantiomer of complex B1 (pathway P3). Accordingly, doubling the concentration of the input (D-IN1), as well as both D-A1 and L-A1 complexes, resulted in near stoichiometric activation of both chiral reporters (Figure 2d). As anticipated, similar results were obtained using L-IN1 in place of D-IN1 (pathway P6, Figure S2). The fluorescent signal associated with LR1 (Cy5) remained unaffected when the reaction (pathway P3) was carried out in either the absence of reporter D-R1 or in the presence of a 2-fold excess of D-R1 relative to L-R1 (Figure S3), further confirming absolute orthogonality between D-OUT1 and L-OUT1 (and DDNA and L-DNA in general). Due to its ability to convert an enantiomerically pure DNA input into a 1:1 mixture of orthogonal D-DNA and L-DNA outputs, we term this circuit a “racemization gate”, the outputs of which offer a general route to parallelization of DNA circuitry or other dynamic DNA devices without a concomitant increase in crosstalk between reaction components. In order to demonstrate the application of Reaction B (Figure 1c) in a DNA circuit, we constructed a simple “chirality OR” gate capable of generating an L-DNA output (L-OUT2) from either a D-DNA (D-IN2) or a LDNA (L-IN2) input having the identical sequence (Figure 3a). Thus, the chirality of the input rather than its sequence represents logic values (Figure 3b). Again, we
exploited a stereospecific reporter complex (L-R2) in order to monitor the progress of the reaction. As shown in Figure 3b, the experimental data exhibited the expected Boolean OR logic; the presence of either D-IN2 or L-IN2 ({1,0} and {0,1}, respectively) gave rise to a fluorescent signal that was at least 20-fold stronger than the maximum response seen in the absence of an input ({0,0}). These data were further verified by gel electrophoresis (Figure S4). To the best of our knowledge, this is the first example of a chirality-based DNA computation.
Figure 3. A heterochiral strand-displacement circuit implemented with Reaction B. (a) Schematic illustration of the chirality OR gate and associated reporter complex (LR2). (b) Fluorescence monitoring (Cy5) of the chirality OR gate. Reaction conditions are identical to those reported in Figure 2d.
The data revealed that the chirality OR gate functioned significantly slower with D-IN2 ({1,0},) than with either input containing L-IN2 ({0,1} and {1,1},) (Figure 3b). This observation is most likely attributed to DNA strand L-OUT2 inducing a left-handed chirality onto the complexed PNA strand (i.e. heteroduplex L-A2 is a lefthanded helix).5a,9 Therefore, the strand-displacement reaction between L-A2 and D-IN2 (but not L-IN2) is expected to incur an energetic penalty due to the inversion of duplex helicity en route to product formation10, reducing the overall reaction rate. Consistent with this explanation, when we inverted the configuration of complex A2, replacing L-A2 with D-A2 (a right-handed helix), the opposite trend in reactivity was observed (Figure S5). Future efforts are needed to more definitively characterize the biophysical and kinetic properties of this unique strand-displacement reaction. Finally, we investigated the ability of Reaction B (Figure 1c) to translate a natural D-RNA input signal into an L-DNA output signal, thereby providing a strategy to autonomously interface endogenous nucleic acids with diagnostic and/or therapeutic nanodevices comprised of bioorthogonal L-DNA.11 The use of L-DNA in this context is preferable over other nuclease resistant
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oligonucleotide analogues (e.g. 2′-O-methyl ribonucleotides12) because L-DNA is less likely to interact nonspecifically with endogenous nucleic acids, a property that we anticipate will significantly increase the performance of intracellular DNA-based devices. The heterochiral strand-displacement reaction depicted in Figure 3a was designed with this application in mind: the input strand (D-IN2) is the DNA analog of microRNA-155, an oncogenic microRNA implicated in cancer development.13 Accordingly, when this circuit was initiated with microRNA-155 (D-INRNA) (Figure 4a), it behaved in the same manner, rapidly generating a fluorescent signal corresponding to production of L-OUT2 (Figure 4b). The reaction occurred slightly faster with the RNA input (DINRNA, 5.13 103 M-1 s-1) than with the DNA input (DIN2, 9.56 102 M-1 s-1) (Figure S6), probably due to increased thermodynamic stability of toehold binding interactions. The circuit also behaved as expected in the presence of excess nonspecific RNA (HeLa cell nuclear RNA), demonstrating the specificity of the heterochiral strand-displacement reaction. All reactions were carried out at 37 °C. Taken together, these data indicate that heterochiral circuitry is compatible with biologically relevant D-RNA inputs, providing a starting point for engineering bioorthogonal L-DNA-based nanodevices that interact with and operate within living cells.
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ily integrated with the majority of preexisting homochiral systems. This provides an opportunity not only to parallelize traditional strand-displacement circuitry, but also to increase the variety of architectures and dynamic behaviors that can be programed into DNA-based nanodevices. In addition, the recent discovery of “crosschiral” aptamers and ribozymes6b,14, both of which interact with nucleic acids of opposing chirality through shape rather than sequence complementarity, may expand the toolkit for engineering heterochiral DNA nanotechnology beyond what is presented here. L-DNA is resistant to both nuclease degradation and off-target interactions with native nucleic acids and proteins.4 Thus, our demonstration of sequence-specific interfacing of a biologically relevant RNA species (microRNA) with L-DNA lays the foundation for integrating endogenous nucleic acid signals with L-DNA-based “biocomputers” capable of performing autonomous diagnostic and therapeutic tasks in living organisms free of obstruction from cellular components.11,15 Success in this area would remove a significant source of design constraints in the burgeoning field of in vivo DNA nanotechnology. Towards this goal, the heterochiral circuit depicted in Figure 4a could easily be employed as a bioorthogonal sensor for oncogenic microRNA-155. Moving forward, it will be important to explore the operation of heterochiral strand-displacement circuitry in live cells. ASSOCIATED CONTENT Supporting Information. Materials and methods. Table S1 and Figures S1–S6. The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author
Figure 4. Heterochiral strand-displacement circuits are capable of interfacing native D-RNA with L-DNA. (a) Schematic illustration of a heterochiral circuit that translates a natural D-RNA input signal into a L-DNA output signal. The components of this circuit, including the reporter complex, are identical to those used in Figure 3a, however, an RNA version of D-IN2 (D-INRNA) was used as the input strand. D-INRNA is microRNA-155. (b) Fluorescence monitoring (Cy5) of the heterochiral circuit in (a). Reaction mixtures contained 500 nM of each circuit component, 300 mM NaCl, and 10 mM Tris (pH 7.6), and were carried out at 37 °C. The asterisk indicates the presence of 0.1 mg/mL HeLa cell nuclear RNA extract.
In summary, we have designed and implemented two novel strand-displacement reactions capable of interfacing the two orthogonal enantiomers of DNA in a sequence-specific manner (Figure 1), thereby establishing chirality as a design parameter for DNA nanotechnology. Because these heterochiral strand-displacement reactions adhere to simple WC base pairing rules, they can be eas-
*
[email protected] Author Contributions
‡These authors contributed equally. Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT The authors are grateful to Dr. Marc Greenberg, Dr. David Horning, Dr. Gerald Joyce, and Dr. Matthew Sheldon for insightful suggestions and comments. This work was supported by the Cancer Prevention and Research Institute of Texas (M1503504) and The Welch Foundation (A1909).
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