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Small Molecule Release and Activation through DNA Computing Kunihiko Morihiro, Nicholas Ankenbruck, Bradley Lukasak, and Alexander Deiters* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States S Supporting Information *

ABSTRACT: DNA-based logic gates can be assembled into computational devices that generate a specific output signal in response to oligonucleotide input patterns. The ability to interface with biological and chemical environments makes DNA computation a promising technology for monitoring cellular systems. However, DNA logic gate circuits typically provide a single-stranded oligonucleotide output, limiting the ability to effect biology. Here, we introduce a novel DNA logic gate design capable of yielding a small molecule output signal. Employing a Staudinger reduction as a trigger for the release and activation of a small molecule fluorophore, we constructed AND and OR logic gates that respond to synthetic microRNA (miRNA) inputs. Connecting the gates in series led to more complex DNA circuits that provided a small molecule output in response to a specific pattern of three different miRNAs. Moreover, our gate design can be readily multiplexed as demonstrated by simultaneous small molecule activation from two independent DNA circuits.

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INTRODUCTION The inception of DNA computation has been widely attributed to Adleman’s work on solving the Hamiltonian path problem using DNA hybridization.1 Since then, several DNA-based circuits have been engineered to act as molecular computation devices by utilizing DNA as inputs, outputs, and logic gates. DNA logic gates have been designed as general computational devices that can mimic the strict control and organization of electronic systems.2 Most commonly, these devices convert oligonucleotide inputs into an output signal via a series of toehold-mediated strand exchange reactions in which toe-holds hybridize to specific regions of the input sequences to facilitate displacement of a single-stranded DNA molecule from logic gate structures. DNA logic gates have been used for increasingly complex applications such as neural networks,3 tic-tac-toe,4 edge detectors,5 and DNA nanoprocessors.6 A key feature of these nucleic acid devices is that they can recognize biological inputs such as DNA and RNA, and recent developments led to interfacing them with protein activation.7 DNA nanostructures have also emerged as compatible with cellular environments.8 Small molecules have been widely used as drugs, dyes, and research tools. Their broad biological function makes them important candidates for direct interfacing with DNA computation circuits. However, DNA logic gates commonly release only an oligonucleotide output. Few oligonucleotide drugs are used in clinical practice; however, interfacing DNA circuits with hundreds of available FDA-approved small molecule drugs would broaden the therapeutic potential of DNA computation. For example, the ability to release a functional small molecule following recognition of microRNA (miRNA) patterns could enable a therapeutic response to a distinct disease biomarker. As a proof-of-concept model, we functionalized synthetic DNA oligonucleotides with small molecule fluorophores, which remain inactive until triggered © 2017 American Chemical Society

by a DNA computation reaction. MiRNA sequences were selected as inputs for the DNA computation events. MiRNAs are small single-stranded noncoding RNA molecules, which regulate gene expression via binding to the 3′ untranslated region of complementary mRNA.9 It has been estimated that more than 60% of protein-coding genes in humans contain miRNA target sites10 and miRNA misregulation has been linked to the development of various human diseases.11 The release and activation of a small molecule through DNA computation of miRNA inputs would enable the development of unique miRNA signature detection and visualization tools and miRNA-based therapeutics. Our approach presented here utilizes a Staudinger reduction as a trigger for the release of small molecules. The Staudinger reduction of an azide by a phosphine exhibits rapid kinetics and a high degree of bioorthogonality, as demonstrated by nucleic acid sensors in test tubes,12 bacterial,13 and mammalian cells.14 To integrate this technology into DNA computation circuits, we designed gate complexes assembled from chemically modified synthetic DNA oligonucleotides (Figure 1). A free phosphine-modified DNA strand is generated as the result of an upstream DNA computation event that initiates a toe-holdmediated strand displacement reaction to form a duplex with a small molecule-azido-modified oligonucleotide. The Staudinger reduction only occurs when a DNA hybridization events places the reaction partners into close proximity. The electrondonating amino group triggers fragmentation of the linker via 1,6-elimination and subsequent release and activation of the small molecule. This design was first validated with two fluorophores: 7-amino-4-methyl coumarin (AMC)15 and 4amino-N-butyl-1,8-naphthalimide (ABNI).16 Their fluorescence Received: July 26, 2017 Published: September 25, 2017 13909

DOI: 10.1021/jacs.7b07831 J. Am. Chem. Soc. 2017, 139, 13909−13915

Article

Journal of the American Chemical Society

Figure 1. Staudinger reduction-mediated small molecule release gate design. (A) The output from an upstream DNA computation event initiates a strand displacement reaction positioning the phosphine P and azide moieties in close proximity. This results in release and activation of a small molecule through a Staudinger reduction and a concomitant 1,6-elimination. Toe-hold regions are shown in red. 2DPBM-DNA is illustrated as a phosphine-modified DNA. (B) The structures and the excitation/emission wavelengths of the fluorophores F used in this study: AMC = 7-amino-4methylcoumarin and ABNI = 4-amino-N-butyl-1,8-naphthalimide.

local concentration of the reaction partners.18 The reaction kinetics of the Staudinger reduction strongly depend on the structure of the phosphine;19 hence, we next optimized the structure of the phosphine-modified DNA. Among the phosphine-modified DNA strands investigated, 2DPBMmodified DNA demonstrated the fastest kinetics of AMC release (Supporting Figure 3). We hypothesize that this is due to the ortho-amide group assisting hydrolysis of the intermediate aza-ylide through neighboring group participation.19a Thus, 2DPBM-modified oligonucleotides were employed in all subsequent experiments. Following optimization of small molecule activation using single-stranded DNA, an AND gate that releases a small molecule after recognition of miR-122 and miR-21 input sequences was constructed on the basis of DNA sequence design concepts reported by Winfree.20 Both miRNAs have been implicated in several human diseases,21 and targeted small molecule release may provide novel diagnostic and therapeutic opportunities. A translator gate (TG122) was used to convert the miR-122 input into a phosphine-modified strand. The miR21 input removes the first gate strand from the AMC-azidomodified AND gate (AND-G21), exposing a new toe-hold, which is recognized by the 2DPBM-modified output from TG122. This results in the release of the final DNA strand and ultimately the activation and release of a small molecule fluorophore (Figure 2). Staudinger reduction only occurs in response to hybridization of the 2DPBM-modified DNA and AMC-azido-modified DNA as a result of strand displacement reactions initiated by both inputs. DNA oligonucleotides with miRNA sequences were used as inputs, in line with previous studies.20 Relative fluorophore output was calculated using standard curves of AMC- or ABNI-azido-modified DNA and free fluorophores as described in the Supporting Information. Measurements were normalized to the highest output signal in each DNA circuit. Maximal AMC release was observed only when both miR-21 and miR-122 sequences were added to the gate complex enabling unambiguous DNA logic gate outputs. However, 0.4-fold background activation was observed with

is greatly reduced by converting their aromatic amino group into a carbamate moiety. The small molecule fluorophores are then activated upon release from the logic gate through carbamate fragmentation and regeneration of the amine. The excitation and emission wavelengths of the two AMC and ABNI fluorophores are sufficiently different to allow for the selective detection of independent small molecule activation.

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RESULTS AND DISCUSSION The fluorophore-azido- and phosphine-modified oligonucleotides were prepared via conjugation reactions between the corresponding NHS-ester and the 5′- or 3′-amino-modified DNA (Supporting Schemes 1 and 2 and Supporting Tables 1− 3). To determine the requirement for hybridization of the modified strands for release of a fluorophore, we tested 3(diphenylphosphino)propanamido (3DPPM)-modified DNA with modified with complementary or noncomplementary AMC-azido-modified DNA oligonucleotides in TE/Mg2+ buffer, which is commonly used for in vitro DNA computation experiments to enhance the stability of DNA duplexes (Supporting Figure 1).17 One equivalent of 3DPPM-DNA combined with complementary AMC-azido-DNA led to small molecule fluorescence activation through Staudinger reduction within 1 h. However, when 3DPPM-modified DNA and scrambled AMC-azido-modified DNA were incubated at equimolar concentrations, significantly reduced reaction kinetics and lower fluorescence were observed. To further determine if the phosphine and azide moieties need to be in close proximity through DNA assembly to trigger small molecule release, we attempted to react AMC-azido-modified DNA with a small molecule, non-DNA-conjugated 2(diphenylphosphino)benzamide (2DPBM) derivative (2DPBM-K, which has better water solubility than 2DPBM) (Supporting Scheme 3). Gratifyingly, we observed that at least a 100-fold excess of 2DPBM-K was required for the release of AMC from the fluorophore-azido-modified DNA (Supporting Figure 2) as compared to the DNA-conjugated phosphine, indicating the importance for DNA-hybridization enhanced 13910


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applicability of the small molecule release gate, we designed a DNA logic gate to respond to miR-122 OR miR-125b. Unlike the AND gate, the miR-122 OR miR-125b gate provides an output in the presence of either or both of the two miRNA inputs. The gate design consists of two 2DPBM-modified translator gates (TG122 and TG125b) and an AMC-azidomodified reporter gate (FG) (Figure 3). The outputs of TG122 and TG125b both have an identical toe-hold region capable of interacting with FG, which is blocked by a complementary DNA strand. When miR-122 or miR-125b is added to the gate circuit, they can hybridize to their respective gate via an exposed toe-hold, releasing the 2DPBM-modified DNA from TG-122 or TG-125b, respectively. The 2DPBM-modified output strands then initiate the same subsequent strand displacement reaction with FG and release an AMC output via Staudinger reduction. As expected, AMC release was detected when either miR-122 or miR-125b was added; however, only a 0.3-fold relative output of AMC was observed in the absence of any input miRNA, providing the expected OR response. The “wiring” of multiple DNA logic gates has enabled the construction of multilayer circuits that constitute more intricate devices.26 To enable small molecule release in response to more complex miRNA input patterns, we designed a small DNA circuit in which an OR and an AND gate were connected in series. This circuit results in the release of a small molecule only when miR-21 and miR-122 or miR-125b are present. MiR-122 or miR-125b hybridize to the corresponding OR gate, converting the input strand into a 2DPBM-modified ssDNA oligonucleotide. Similar to the AND gate described above, the 2DPBM-modified strand can only hybridize to the gate after miR-21 displaces the top strand leading to exposure of a new toe-hold. The resultant strand displacement reaction triggers release and activation of the AMC output (Figure 4). Consistent with the truth table for the circuit, either miR-122 and miR-21 inputs or miR-125b and miR-21 inputs yielded a “true” output. In the absence of miR-21 and/or both miR-122 and miR-125b, minimal AMC release was observed corresponding to a “false” output. The OR gate delivers two-times more 2DPBM-modified DNA in the presence of both miR-122

Figure 2. (A) Electronic symbol for the miR-21 AND miR-122 gate. (B) Simplified schematic of the small molecule release from the miR21 AND miR-122 gate. Toe-hold regions are shown in green, red, and blue. (C) The structure of the phosphine, 2DPBM, used in these experiments. (D) AMC fluorescence is shown for addition of both miR-21 and miR-122 in different combinations to the gate complex. The mixture of TG122 and AND-G21 was incubated at 37 °C, and the fluorescence intensity of AMC was measured after 1 h. Three independent experiments were averaged, and the error bars represent standard deviations.

addition of only miR-21. We hypothesize that AT-rich regions within the second toe-hold sequence and at the end of the TG122 duplex may partly dissociate (or fray), enabling background activation.22 The current design may be improved by the introduction of “clamps” to the toe-hold region,23 single base mutations,24 or additional GC pairs at the duplex termini.25 Additionally, some background activation may have occurred because the gate duplexes were not gel purified, which led to undesired phosphine oxidation. This initial characterization revealed that the novel Staudinger reduction-based gate is functional and enables the selective release of a small molecule output. The miR-21 AND miR-122 gate requires both miRNA inputs to generate the small molecule output. To further evaluate the

Figure 3. (A) Electronic symbol for the miR-122 OR miR-125b gate. (B) Simplified schematic of the small molecule release from the miR-122 OR miR-125b gate. Toe-hold regions are shown in green, purple, and blue. (C) AMC fluorescence is shown for addition of either miR-122 or miR-125b in different combinations to the gate complex. The mixture of TG122, TG125b, and FG was incubated at 37 °C, and the fluorescence intensities of AMC were measured after 1 h. Three independent experiments were averaged, and the error bars represent standard deviations. 13911


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Figure 4. (A) Electronic circuit representing (miR-122 OR miR-125b) AND miR-21. (B) Simplified schematic of the small molecule release from the (miR-122 OR miR-125b) AND miR-21 gate. Toe-hold regions are shown in green, purple, red, and blue. (C) AMC fluorescence is shown for addition of both miR-122 and miR-21 or miR-125b and miR-21 in different combinations to the gate complex. The mixture of TG122, TG125b, and AND-G21 was incubated at 37 °C, and the fluorescence intensities of AMC were measured after 1 h. Three independent experiments were averaged, and the error bars represent the standard deviations.

Figure 5. (A) Electronic circuits representing (miR-122 OR miR-125b) AND miR-21 and (miR-15a OR miR-143) AND miR-10b. (B) AMC fluorescence and ABNI fluorescence is shown for addition of miR-21, miR-122, miR-125b, miR15a, miR-10b, and miR-143 in different combinations to the two gate complexes. The mixture of TG122, TG125b, AND-G21, TG15a, TG143, AND-G10b, and inputs was incubated at 37 °C, and the fluorescence intensities of AMC and ABNI were measured after 1 h. Three independent experiments were averaged, and the error bars represent standard deviations. 13912


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approach in the fields of synthetic biology and nanotechnology; however, translating single-stranded oligonucleotide outputs into triggers for biological and/or chemical events has been limited. We are addressing the restrictions that this imposes on the design and application of DNA computation through new AND and OR gates that generate small molecule outputs in response to miRNA input patterns. These systems are functional in the context of a small DNA-based circuit (3 inputs) and were quickly expanded into more complex systems through multiplexing (6 inputs). Because of the importance in understanding their regulation and function, several strategies have been previously employed for the triggering of DNA nanodevices by miRNAs in cells and animals. For example, sequence-specific fluorescent peptide nucleic acid probes were utilized for imaging miRNAs in mammalian cells14b and zebrafish embryos.37 We and other groups previously employed DNA-based devices in the detection of miRNA expression in mammalian cells.38 Signal amplification strategies utilizing DNA self-assembly allowed for detection of cellular miRNAs.39 In addition to miRNAs, engineered DNA devices for the detection of endogenous messengerRNAs (mRNAs) in human cells have been reported.40 However, current DNA logic devices capable of recognizing complex RNA input patterns beyond single sequences only release single-stranded oligonucleotides as the eventual outputs.20,41 As compared to nucleic acids or proteins as outputs, small molecule outputs can be freely synthesized, expanding the utility of highly modular DNA computing approaches.

and miR-125b, which may contribute to the higher small molecule output levels in the presence of all three inputs. As expected, addition of all three inputs led to an increase in AMC release and activation as compared to combinations of miR-122 or miR-125b and miR-21. Additionally, only minor fluorescence was detected for other variations of the miRNA inputs. To demonstrate the ability to orthogonally release two different small molecules in response to upstream DNA computation events, without any crosstalk between the two circuits, we designed a second logic gate circuit that could be activated independently of the initial gate design to provide a second unique small molecule output. We combined the miR21 AND (miR-122 OR miR-125b) gate with a newly designed miR-10b AND (miR-15a OR miR-143) gate, which would react independently to provide output 1 (AMC fluorophore) or output 2 (ABNI fluorophore) in response to a specific combination of miRNA inputs.27 We tested fluorophore release from the gates through the addition of several permutations of the six miRNAs in the same test tube. As expected, AMC release was observed in the presence of both miR-122 or miR125b and miR-21, while ABNI was released in the presence of both miR-15a or miR-143 and miR-10b (Figure 5). Because the total concentration of phosphine-modified DNA contained in this multiplexed gate solution was 800 nM, we hypothesize that the higher background signal arises from the high phosphineDNA concentration inducing Staudinger reduction independent of DNA hybridization. This is consistent with elevated concentrations of small molecule phosphine being capable of activating AMC-azido-modified DNA in the absence of a DNA hybridization reaction (Supporting Figure 2). This is a trade-off that was made in demonstrating circuit multiplexing. More integrated circuit designs that do not utilize multiplexing and minimize the number of phosphine-DNA gates would most likely show reduced background levels but would also require a more complicated sequence design. Using the circuits and thereby releasing small molecules at lower concentrations represents a separate solution. For example, IC50’s of cytotoxic drugs below 100 nM are quite common.28 The ability to release small molecule outputs (e.g., fluorophores or therapeutic agents) in response to nucleic acid input patterns has implications in the detection and treatment of diseases. For example, several drugs have been shown to be deactivated by conversion of an amino group to a carbamate, including doxorubicin,29 melphalan,30 gemcitabine,31 tamoxifen,32 nitrogen mustard,33 auristatin E,34 and anisomycin,35 making these biologically active small molecules amenable to interfacing with our DNA logic gate design. As compared to other oligonucleotide sensing applications, the requirement of proximity of the two reaction partners to trigger small molecule activation ensures that enzymatic degradation of the DNA will not lead to a nonspecific signal. Additionally, the irreversible nature of the Staudinger reduction and small molecule release constitutes a thermodynamic sink that is difficult to realize if only DNA hybridization is utilized. Furthermore, the potential to release precipitating dye molecules, such as X-gal,36 could enable a method for a simple visual readout of miRNA pattern detection and may provide staining of cells and tissues without the need for fluorescence.

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EXPERIMENTAL SECTION

Modified DNA Preparation. Modified DNAs were prepared by amide bond formation between the NHS-ester and the 5′- or 3′amino-modified DNA. The details of the chemical synthesis of the NHS-esters are included in the Supporting Information. Aminomodified DNAs were purchased HPLC-purified from IDT. For the preparation of the fluorophore-azido-modified DNA, 10 nmol of the 3′-amino-modified DNA and 400 nmol of the corresponding NHSester in 100 mM sodium bicarbonate solution (H2O:DMF = 3:2, 200 μL) were reacted for 4 h at room temperature. The reaction product was collected by ethanol precipitation and subsequently purified by reverse-phase HPLC (5−50% acetonitrile/100 mM triethylammonium acetate gradient). For the preparation of the phosphine-modified DNA, 6 nmol of the 5′-amino-modified DNA and 240 nmol of the corresponding NHS-ester in 100 mM sodium bicarbonate solution (H2O:DMF = 7:3, 100 μL) were reacted for 4 h at room temperature. The reaction product was directly purified by reverse-phase HPLC (5−40% acetonitrile/10 mM triethylammonium acetate gradient). All DNA structures were confirmed by ESI−MS analysis. Logic Gate Preparation. Unmodified DNAs were purchased from Sigma. Gate complexes were formed by mixing final concentrations of 2 μM of modified DNA and 4 μM of unmodified DNA in TE/Mg2+ buffer (10 mM tris-HCl, 100 mM ethylenediaminetetraacetic acid (EDTA), and 12.5 mM MgCl2). Gate formations were confirmed by native polyacrylamide gel electrophoresis (PAGE). All gate complexes were directly used for the following experiments without further purification. Gate Functional Examination. Fluorescence was measured on a Tecan M1000 plate reader in black 96- (Costar) or 384-well plates (Fisher). All gates were analyzed at 200 nM with 800 nM miRNA inputs in 100 or 50 μL of TE/Mg2+ buffer (10 mM tris-HCl, 100 mM ethylenediaminetetraacetic acid (EDTA), and 12.5 mM MgCl2; for cost reasons, DNA oligonucleotides with the miRNA sequences were used as inputs) at 37 °C. Relative concentrations of released fluorophores were calculated using standard curves of the AMC- or ABNI-azido-modified DNA and the fluorophore (Supporting Figure

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CONCLUSIONS We demonstrated small molecule release through a variety of DNA logic gate circuits in response to synthetic oligonucleotide inputs. DNA computation has emerged as a highly versatile 13913


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4) and then normalized to the highest output signal. Triplicate experiments were performed for each condition.

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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.7b07831. Modified oligonucleotide synthesis protocols, NMR spectra of new compounds, oligonucleotide sequences, and supporting figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Alexander Deiters: 0000-0003-0234-9209 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Science Foundation (CCF-1617041). K.M. is grateful for a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Research Abroad. We thank Alexander Prokup for assistance with DNA logic gate design.

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REFERENCES

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