Dynamic Control of AptamerâLigand Activity Using Strand

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Letter

Dynamic control of aptamer-ligand activity using strand displacement reactions Jonathan Lloyd, Claire H. Tran, Krishen Wadhwani, Christian Cuba Samaniego, Hari K. K. Subramanian, and Elisa Franco ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00277 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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ACS Synthetic Biology

Dynamic control of aptamer-ligand activity using strand displacement reactions Jonathan Lloyd,†,¶ Claire H. Tran,†,¶ Krishen Wadhwani,† Christian Cuba Samaniego,‡ Hari K. K. Subramanian,‡ and Elisa Franco∗,‡ †Bioengineering, University of California at Riverside, Riverside, CA 92521 ‡Mechanical Engineering, University of California at Riverside, Riverside, CA 92521 ¶Contributed equally to this work E-mail: [email protected]

Abstract Nucleic acid aptamers are an expandable toolkit of sensors and regulators. To employ aptamer regulators within non-equilibrium molecular networks, the aptamer-ligand interactions should be tunable over time, so that functions within a given system can be activated or suppressed on demand. This is accomplished through complementary sequences to aptamers, which achieve programmable aptamer-ligand dissociation by displacing the aptamer from the ligand. We demonstrate the effectiveness of our simple approach on light-up aptamers as well as on aptamers inhibiting viral RNA polymerases, dynamically controlling the functionality of the aptamer-ligand complex. Mathematical models allow us to obtain estimates for the aptamer displacement kinetics. Our results suggest that aptamers, paired with their complement, could be used to build dynamic nucleic acid networks with direct control over 1 ACS Paragon Plus Environment


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a variety of aptamer-controllable enzymes and their downstream pathways. Keywords: Aptamer, strand displacement, dynamics, synthetic biology. Programming RNA molecules to redirect cellular functions is increasingly simple and fast, due to the advances made in two complementary approaches to RNA sequence design: computational tools mapping RNA sequence and secondary structure (1 ), and in vitro evolution techniques enabling the identification of RNA sequences with affinity for desired ligands (2 , 3 ). In particular, SELEX allows the selection of short nucleic acid sequences, called aptamers, that can target many organic or inorganic ligands (2 , 3 ). Synthetic aptamers have been primarily used for biosensing applications (4 , 5 ), however their role as transcriptional or translational regulators is gaining increasing attention (6 ). Recent screening of the E. coli genome combined with SELEX uncovered the existence of a wide class of natural RNA polymerase-binding aptamers that can control transcription by favoring termination (7 ). Thus, aptamers have a significant potential to be employed for dynamic control of biomolecular processes both in vitro and in vivo. Aptamers working as tunable regulators in dynamic molecular networks should exhibit reversible, temporally controllable interactions with their target ligand, enabling activation or deactivation of the ligand functionality on demand and within complex feedback loops. To achieve this goal, a promising approach is to use aptamers in conjunction with rational design techniques developed from dynamic nucleic acid nanotechnology (8 ). These techniques rely on strand displacement and toehold-mediated branch migration (8 , 9 ), and are at the basis of several biomolecular implementations of dynamic circuits such as controllers, bistable networks, and oscillators (10 –14 ). So far, strand displacement networks have been coupled with aptamers for two primary purposes: the generation of amplified molecular signals to detect very small amounts of ligand (15 , 16 ), and logic processing of multiple detection events from distinct aptamers (17 –19 ). In these applications, aptamers bind to their ligand reaching a desired aptamer-ligand equilibrium concentration, which is not influenced by 2 ACS Paragon Plus Environment

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subsequent changes in the downstream or upstream network. Here, we describe a simple method to make aptamer-ligand interactions reversible using strand displacement. We focus on aptamers that activate or deactivate a specific functionality upon binding to their ligand: thus, aptamer-ligand association or dissociation alters a relevant property of the sample or a pathway output in a quantifiable manner. We achieve aptamer displacement from its ligand by using an anti-sense nucleic acid strand, that is partially or fully complementary to the aptamer. For convenience, throughout the manuscript we name these anti-sense strands “kleptamers” (from the Greek word κλ´ επτης, kléptes, thief). Kleptamers generalize the idea of aptamer “antidotes” - oligonucleotides or polymers designed to displace and neutralize aptamers selected against blood coagulation factors (20 , 21 ). First, we demonstrate kleptamers working with light-up RNA aptamers, that are becoming increasingly popular as fluorescent reporters. We designed and tested kleptamers for Spinach (22 ), Malachite Green (23 ), and Mango aptamers (24 ), showing that near complete displacement is achieved on the order of minutes, and introduction of toehold domains is not required. Next, we modulate in vitro transcription using aptamer-kleptamer pairs for T7 and SP6 bacteriophage RNA polymerases (RNAP): RNA aptamers inhibiting these enzymes (25 , 26 ) can be effectively and rapidly displaced, tuning the fraction of active enzyme and thus the measured transcription rate of a reporter Broccoli aptamer. We demonstrate aptamer displacement using both equilibrium and dynamic (cotranscriptional) experiments. We complement our experiments with simple ordinary differential equation (ODE) models, which allow us to estimate aptamer-kleptamer kinetic rates comparable to those measured in other nucleic acid reaction networks.

Results Control of reporter fluorescence using strand displacement. Our goal is to demon-

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strate that strand displacement can be used to regulate aptamer-ligand binding, and thus control the function (activity) of the aptamer-ligand complex. We illustrate this idea using “light-up” RNA aptamers, which are RNA sequences selected to bind to small molecules and switch on their fluorescence (22 –24 , 27 –29 ). We start with the Broccoli aptamer (22 ), which binds to (Z)-4-(3,5- difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)one (DFHBI), a structural mimic to the Green Fluorescent Protein fluorophore (4-hydroxybenzylidene-imidazolinone, HBI). DFHBI becomes fluorescent only when bound to the aptamer (Fig. 1 A). To reverse the aptamer-ligand binding, and thus modulate the fraction of aptamer-ligand complex, we designed DNA aptamer-displacing sequences, or kleptamers, that are complementary (or partly complementary) to the Broccoli aptamer and are expected to displace it from its target, decreasing the measured fluorescence of the sample. Starting from a solution containing aptamer-DFHBI complex at equilibrium, we added increasing DNA kleptamer amounts and measured the fluorescence of the solution, obtaining kinetic measurements of strand displacement (Fig. S5 A). We considered three kleptamer variants: one fully complementary to the aptamer (variant Kbf), one complementary to the top stem region (variant Kb1), and one complementary to the bulge region that serves as a binding pocket for DFHBI (variant Kb2). The expected secondary structure of the aptamer and the binding region of the variants are shown in Fig. 1 B (left) and SI Fig. S1. A change in fluorescence was measured only upon addition of Kbf and Kb2, and the reaction reaches steady state on a timescale of about 3-4 minutes, as shown in Fig. 1 B (middle). In contrast, variant Kb1, targeting the terminal stem loop, did not produce any visible change in fluorescence, presumably because it does not successfully bind to the aptamer or it does not displace the dye from its binding pocket. A full complement RNA kleptamer binds to the aptamer and displaces the ligand as efficiently as the full complement DNA kleptamer, as shown in Fig. 1 B, right (hybridization of full kleptamer and aptamer was verified via native gel electrophoresis, Fig. S5 B). We then obtained the curve in Fig. 1 C, which maps 5 ACS Paragon Plus Environment


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full kleptamer (Kbf) concentration and fraction of aptamer-DFHBI complex. This map is consistent with stoichiometric binding of aptamer and kleptamer. Assuming all aptamer is initially bound to DFHBI, we estimated the rate at which the kleptamer displaces the aptamer using a simple model reaction: k+

− ⇀ B · DF HBI + Kbf − ↽ − − B · Kbf + DF HBI, −

(1)

k

where B is the Broccoli aptamer, and Kbf is the full kleptamer. With MATLAB, we integrated the corresponding ODEs starting from initial conditions consistent with the experiments, and we used them to fit the equilibrium curve in Fig. 1 C: we obtained an estimated forward rate k + = 8.9 · 105 /M/s. This estimate is on the same order of magnitude of toehold-mediated strand displacement reactions (8 , 10 ), suggesting that the 3’ end region of the aptamer (Fig. 1 B, left and Fig. S1) may act as a natural toehold promoting displacement. However, the shorter variant Kb2 achieves displacement kinetics similar to the full kleptamer Kbf (Fig. 1 B, middle), indicating that unpaired nucleotides near the dye binding pocket might promote fast displacement. We obtained similar results with a full kleptamer for the light-up aptamer Malachite Green (23 , 30 ) (Fig. S2 C): the equilibrium curve mapping kleptamer concentration and fraction of aptamer-Malachite Green complex is shown in Fig. S6. Finally, we asked if displacement could also be achieved with the Mango aptamer (24 ) (Fig. S2 A), a sequence that binds to Thiazole Orange-Biotin dye (TO1-Biotin); the dissociation constant of Mango from TO1-Biotin is 3.2 nM, as reported by Dolgosheina et al. (24 ), at least one order of magnitude lower than the dissociation rates of Spinach aptamer (300-540 nM) and Malachite Green aptamer (117 nM) from their targets (24 ). We achieved displacement of Mango aptamer with its full kleptamer (Fig. S7); introduction of a toehold domain (Fig. S2 B) on the aptamer resulted in a displacement reaction only moderately faster (Fig. S7), which


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may be due to the aforementioned low aptamer-dye dissociation rate, or to the secondary structure of the aptamer itself. Regulating RNA polymerase activity via DNA kleptamer displacement of inhibiting aptamers. Next, we tested if strand displacement of aptamers from their target could be used to control a biological pathway. We considered in vitro transcription with bacteriophage RNA polymerases (RNAPs), which are widely used in synthetic biology applications (31 , 32 ). We adopted two RNA aptamers selected to inhibit transcription activity of T7 and SP6 RNA polymerases (25 , 26 , 33 ). We expect that addition of kleptamers displacing the RNAP-inhibiting aptamer from the (inactive) aptamer-RNAP complex will restore RNAP transcription activity (Fig. 2 A). To monitor and quantify the effectiveness of kleptamers we used the Broccoli fluorescent aptamer as a reporter, controlling its transcription from a synthetic template including either the T7 or SP6 bacteriophage promoter. From normalized kinetic data of Broccoli transcription, we estimated the fraction of active RNA polymerase as a function of aptamer and kleptamer concentration, and we built the curves shown in Figure 2 (the normalization procedure is reported in SI Section 5). We first considered the T7 RNAP-binding aptamer (25 , 33 ), and we designed a DNA kleptamer that is complementary to the aptamer (Fig. S3 A). Using our Broccoli reporter assay, we first verified the inhibitory capacity of the T7 aptamer: T7 RNAP was separately incubated with T7 aptamer at different concentrations, resulting in a mix of active and inactive enzyme that depends on the relative concentration of aptamer and polymerase. This mix was then used for in vitro transcription of Broccoli reporter. From the kinetic curves (Fig. S8), we estimated the fraction of active T7 RNAP, plotted in 2 B (left) as a function of aptamer concentration. Then, we tested the capacity of the kleptamer molecule to restore transcription by adding different concentrations of kleptamer to a sample of fully inhibited T7 RNAP (T7 RNAP-aptamer mix); the resulting mix of active and inactive enzyme for Broccoli transcription (the proportion of active enzyme depends on the relative 7 ACS Paragon Plus Environment


DFHBI

A

Active RNA polymerase

Broccoli aptamer Reporter Template

Aptamer

Kleptamer

Transcription off

Waste

B

Transcription on


Inactive RNA polymerase T7 RNA polymerase: inhibition

T7 RNA polymerase: activation

1.2 1

Fraction ofa.u. active T7


1 0.8 0.6 0.4 0.2 0

0

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800

0.8 0.6 0.4 0.2 0

1000

0

Aptamer concentration (nM)

C

SP6 RNA polymerase: inhibition

400

600

800

1000

1500 1200

SP6 RNA polymerase: activation 1

Fraction ofa.u. active SP6

0.8 0.6 0.4 0.2 0

200

Kleptamer concentration (nM)

1


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0.8 0.6 0.4 0.2 0

0

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1000


0

200

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600

800

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1200

Kleptamer concentration (nM)

Figure 2: Aptamers and kleptamers can be used to modulate the activity of bacteriophage T7 and SP6 RNAP. A: We used RNAP-inhibiting aptamers in conjunction with DNA kleptamers to turn on or off transcription of Broccoli reporter. B and C: Curves showing the fraction of active RNAP (either T7 or SP6) as a function of aptamer or kleptamer concentration. Equilibrium data points were obtained by normalizing kinetic transcription curves reported in SI Section 5. Solid lines represent predictions of an ODE model derived from Equations (2) (SI Section 6).


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amount of aptamer and kleptamer) was then used to control transcription of Broccoli reporter (Fig. S9). Figure 2 B, right, shows the normalized active enzyme fraction as a function of kleptamer concentration: the kleptamer successfully displaces the inhibiting aptamer, reactivating transcription. We further demonstrated that transcription via SP6 RNA polymerase can be similarly controlled using an aptamer-kleptamer pair. The tested SP6 kleptamer, showed in SI Fig. S3 B, targets primarily the bulge of the expected SP6 binding pocket, and is not complementary to the full aptamer sequence. The Broccoli reporter assays (Figs. S10 and S11) were used to estimate the fraction of active enzyme in the presence of aptamer and/or kleptamer. After characterizing the effectiveness of the inhibiting aptamer (26 ) as shown in 2 C, left, we verified that kleptamer-mediated displacement of the aptamer restores transcription, as show in 2 C, right. To test our ability to quantitatively describe the behavior these aptamer-kleptamer pairs, we built an ordinary differential equation (ODE) model (SI Section 6) that captures reactions of aptamer-enzyme binding, kleptamer-mediated displacement and titration of aptamer, and transcription. The aptamer (A), kleptamer (K), and RNAP (E) interactions were modeled using the following chemical reactions: α+

AE, A+E ⇋ − α

κ+

E + AK, AE + K ⇋ − κ

ν

A + K → AK,

(2)

from which we derived ODEs using the law of mass action. We estimated the reaction rates above, as well as the total concentration of RNAP, by fitting the transcription data points shown in Fig. 2. These ODEs were used to generate the equilibrium curves (solid lines) in Fig. 2 B and C. The Broccoli reporter concentration was estimated by measuring, in a separate series of experiments, the fluorescence of known concentrations of gel extracted Broccoli in the presence of excess amount of DFHBI (SI Section 3.5) and of an additional



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reference TAMRA-labeled oligonucleotide. We estimated forward rates α+ and κ+ , and titration rate ν in the range of 103 –105 /M/s for both T7 and SP6 aptamer-kleptamer systems (see Table 1 and SI Tables S1 and S2 for additional details), which suggest that the kinetics of our aptamer binding and kleptamer displacement are on the same order of magnitude as DNA/RNA hybridization and displacement (8 ).

Table 1: Aptamer and kleptamer system fitted rates for reactions (2) Parameter

Description

T7 RNAP

SP6 RNAP

α+ (/M/s)

Binding rate of aptamer and RNAP

1.4·103

1.9·105

KD (nM)

Dissociation rate

165.3

59.5

κ+ (/M/s)

Binding rate K and AE

1.8·103

0.4·103

ν (/M/s)

Titration rate A/K

1.4·104

9·103

KD,k (nM)

Dissociation rate AK

0.52

0.47

Dynamic control of transcription using aptamers and RNA kleptamers. To show that aptamers and kleptamers have the potential to be used for dynamic control of molecular pathways, we designed two in vitro artificial reaction networks where the activity of SP6 RNAP is regulated cotranscriptionally with an inhibiting aptamer-kleptamer pair (Fig. 3 A and B). We designed two synthetic templates to transcribe RNA aptamers and kleptamers: the first template, whose transcription is controlled by the T7 promoter, produces an RNA regulator (either aptamer or kleptamer) for SP6 RNAP and thereby controls transcription of the second template, which produces Broccoli reporter downstream of the SP6 promoter. First, we showed that the T7-controlled template can cotrascriptionally inhibit transcription of the SP6-controlled template by producing the SP6 RNAP aptamer (Fig. 3 A). Transcription of the SP6-transcribed Broccoli reporter is rapidly turned off as soon as the T7-controlled template (producing inhibiting aptamer) is added to the reaction mix. Then, the T7-controlled template was designed to produce the SP6 RNA kleptamer se10 ACS Paragon Plus Environment

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quence: inhibited SP6 RNA polymerase (bound to its aptamer) is not expected to transcribe reporter RNA, however in the presence of kleptamer-producing template and T7 RNA polymerase, SP6 RNA polymerase should recover its activity as illustrated in 3 C. Fluorescence kinetic data shown in 3 D indicate that the pathway is performing as expected: reporter production is triggered with a time delay that depends on the concentration of kleptamer producing template. The simple mathematical model derived from reactions (2) was updated to include transcription of aptamer or kleptamer as described in SI Section 6. The rates estimated by fitting the experiments in Fig. 2 B and C could predict the behavior of cotranscriptional experiments; the model predictions are shown as dashed lines in Fig. 3. For these predictions, only the transcription rates of reporter and aptamer/kleptamer in Figures 3 B and D were fitted to control experiments to reflect the fact that distinct enzyme batches (having different activity) were used in each of these assays (see SI Tables S3 and S4).

Discussion and conclusions We have presented a simple strategy to achieve dynamic control of aptamer-ligand function by using strand displacement. The strategy relies on aptamer-complementary nucleic acid strands, or kleptamers, which displace the aptamer from the aptamer-ligand complex. We tested our approach on three light-up aptamers, and two inhibiting aptamers that target bacteriophage RNA polymerases. We built and fitted simple ODE models capturing the relevant reactions in our systems, and found that the estimated reaction kinetic rates of aptamer-ligand and aptamer-kleptamer occur on a timescale comparable to those in nucleic acid networks (8 ). We demonstrated that RNAP aptamer displacement is achieved not only with DNA or purified RNA kleptamers, but also with cotranscriptionally produced RNA kleptamers. RNAP-targeting aptamer-kleptamer pairs can be used to build elementary



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dynamic regulatory modules for activation or inhibition of RNA transcription. Our results suggest that kleptamer complementarity to the aptamer can be partial. Partially complementary kleptamers may have several advantages, including minimal secondary structure, reduced crosstalk with other nucleic acids present in the system, and lower cost. Kleptamers partially complementary to aptamers should target a number of unpaired nucleotides that is sufficient to initiate strand displacement. These unpaired nucleotides may be identified at the 3’ or 5’ end of the aptamer, or may be included in bulge regions of the aptamer that are (presumably) weakly bound to the ligand. This conclusion is supported also by earlier work, where a strand was designed to titrate and displace a Malachite Green aptamer within a circuit achieving perfect adaptation and fold-change detection (34 ): in this circuit, a 5-nucleotide long unpaired domain at the 5’ end of the aptamer was sufficient to promote disassociation of the aptamer-dye complex with a short complementary strand. In our case, a fully complementary kleptamer achieves the same result in the absence of a toehold (Fig. S2 C), indicating that the bulge regions of the aptamer may be sufficient to initiate displacement. Kleptamer-mediated displacement may not be possible in cases where the aptamer-ligand affinity is very strong (35 ). The idea of displacing aptamers from their target has been applied before in the context of controlling blood coagulation factors, and was achieved using an oligonucleotide (36 ) or polymer molecule (21 ) termed antidote. After selection of aptamers with high affinity against specific coagulation factors, RNA antidotes were designed to obtain fast and nearly complete aptamer displacement, neutralizing the aptamer anti-coagulant function both in vitro and in vivo upon injection (20 , 36 , 37 ). As in our case, antidotes need not be fully complementary to the aptamer (36 ). Our work generalizes the antidote idea, and demonstrates that molecules displacing aptamers can be used in the context of reversing aptamer sensing as well as aptamer-based cotranscriptional control of synthetic regulatory networks. The formation of kleptamer-aptamer complexes in our experiments is in practice an ir13 ACS Paragon Plus Environment


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reversible reaction with dissociation rate in the order of fractions of a nanomolar. However, kleptamer-aptamer binding could be made reversible by including a toehold in the kleptamer (9 ): the toehold would enable displacement of the kleptamer by a complementary oligonucleotide and cause release of the aptamer, which could, in turn, bind its ligand restoring functionality. Reversing the kleptamer action could be useful in the context of building molecular dynamic networks. Upstream oligonucleotides displacing the kleptamer would have to be designed with care to avoid the inclusion of aptamer functional domains and spurious interactions with the ligand of interest: this may be done using appropriate sequence mismatches, or by using kleptamers that are only partially complementary to the aptamer. As shown in our experiments, partially complementary kleptamers may be as efficient as fully complementary ones. Regulation of aptamer-ligand activity using strand displacement could be applied in a variety of synthetic biology applications. For instance, aptamer-kleptamer pairs could be used to achieve ultrasensitive responses in aptamer-controlled pathways, where the aptamer concentration would serve as a tunable threshold (38 ). In the context of feedback loop circuits, tunable ultrasensitive elements built with nucleic acids could promote the emergence of bistability or oscillations (39 ). Initial computational studies indicate that the particular RNAP aptamer-kleptamer considered here are suited to build non-equilibrium networks (40 –43 ) which may be highly robust due to the ultrasensitive nature of stoichiometric aptamer regulation (44 ). The success of aptamer displacement in therapeutic applications in vivo (20 ) suggest that aptamer-kleptamer pairs could be immediately used for dynamic regulation of cellular functions.

1

Materials and methods

Further details on materials and methods can be found in the SI file.


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1.1

Oligonucleotides and enzymes

DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). All strands were resuspended in nuclease free water, quantitated by UV absorbance at 260 nm using a Thermo Scientific Nanodrop 2000c Spectrophotometer, and stored at -20◦ C. RNA strands were transcribed and gel extracted in house. SP6 RNA Polymerase and T7 RNA Polymerase (200 U/µL) were both purchased from CellScript (respectively Cat. No. C-S6300K and Cat. No. C-T7300K).

1.2

Oligonucleotide sequences

Sequences are reported in Section 1 of the SI file.

1.3

Strand displacement and transcription

Strand displacement as well as transcription experiments were conducted suspending oligonucleotides or templates in transcription mix, which was prepared with 2 mM NTPs, 1X transcription buffer, nanopure water, and a TAMRA-labeled reference oligonucleotide dye at 100 nM. Transcription mix was used to collect consistent raw fluorescence measurements, which were then converted to estimated concentrations of RNA according to the protocol described in SI Section 3.5. DFHBI dye was used at 15 µM. All transcription experiments were conducted using 300 units of RNA polymerase at 30◦ C.

1.4

Fluorescence data acquisition

Fluorescent measurements were done using a HORIBA Fluorolog-3 spectrophotometer. Excitation and emission wavelength for the dyes were: TAMRA ex/em = 445/575 nm; DFHBI ex/em = 469/501 nm, Malachite Green ex/em = 630/655 nm. The sample temperature was



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set to be 30◦ C for the entire duration of each fluorescence experiment. Sample temperature was equilibrated for 10 minutes before starting the reactions.

1.5

Numerical simulations

Numerical simulations were executed using MATLAB (The MathWorks). Ordinary differential equations were integrated with in house MATLAB scripts using the ode23 routine. Data fitting was performed using the fmincon routine. Details on the data fitting procedure are in Section 6 of the SI file.

Acknowledgement This research was supported in part by the National Science Foundation through grant CMMI-1266402, which paid for most reagents, supplies, and salary to JL, CHT, KW, and CCS; HKKS was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award Number DE-SC0010595. We thank Peter Unrau and Sunny Jeng for their kind gift of Thiazole Orange-Biotin dye. We thank Alessandro Porchetta and Arun Richard Chandrasekaran for useful comments on the manuscript.

Supporting Information Available Detailed information on materials, experimental methods, numerical simulations and additional references is available in the Supplementary Information file. This information is available free of charge via the Internet at http://pubs.acs.org/.


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9. Yurke, B., and Mills, A. P. (2003) Using DNA to Power Nanostructures. Genetic Programming and Evolvable Machines 4, 111–122. 10. Kim, J., White, K. S., and Winfree, E. (2006) Construction of an in vitro bistable circuit from synthetic transcriptional switches. Molecular Systems Biology 1, 68. 11. Franco, E., Friedrichs, E., Kim, J., Jungmann, R., Murray, R., Winfree, E., and Simmel, F. C. (2011) Timing molecular motion and production with a synthetic transcriptional clock. Proceedings of the National Academy of Sciences 108, E784–E793. 12. Montagne, K., Plasson, R., Sakai, Y., Fujii, T., and Rondelez, Y. (2011) Programming an in vitro DNA oscillator using a molecular networking strategy. Molecular Systems Biology 7 . 13. Franco, E., Giordano, G., Forsberg, P.-O., and Murray, R. M. (2014) Negative autoregulation matches production and demand in synthetic transcriptional networks. ACS synthetic biology 3, 589–599. 14. Chen, Y.-J., Dalchau, N., Srinivas, N., Phillips, A., Cardelli, L., Soloveichik, D., and Seelig, G. (2013) Programmable chemical controllers made from DNA. Nature nanotechnology 8, 755–762. 15. Dirks, R. M., and Pierce, N. A. (2004) Triggered amplification by hybridization chain reaction. Proceedings of the National Academy of Sciences of the United States of America 101, 15275–15278. 16. Jiang, Y., Li, B., Milligan, J. N., Bhadra, S., and Ellington, A. D. (2013) Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly. Journal of the American Chemical Society 135, 7430–7433.


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17. You, M., Zhu, G., Chen, T., Donovan, M. J., and Tan, W. (2014) Programmable and multiparameter DNA-based logic platform for cancer recognition and targeted therapy. Journal of the American Chemical Society 137, 667–674. 18. Rudchenko, M., Taylor, S., Pallavi, P., Dechkovskaia, A., Khan, S., Butler Jr, V. P., Rudchenko, S., and Stojanovic, M. N. (2013) Autonomous molecular cascades for evaluation of cell surfaces. Nature nanotechnology 8, 580–586. 19. Han, D., Zhu, Z., Wu, C., Peng, L., Zhou, L., Gulbakan, B., Zhu, G., Williams, K. R., and Tan, W. (2012) A logical molecular circuit for programmable and autonomous regulation of protein activity using DNA aptamer–protein interactions. Journal of the American Chemical Society 134, 20797–20804. 20. Rusconi, C. P., Roberts, J. D., Pitoc, G. A., Nimjee, S. M., White, R. R., Quick, G., Scardino, E., Fay, W. P., and Sullenger, B. A. (2004) Antidote-mediated control of an anticoagulant aptamer in vivo. Nature biotechnology 22, 1423. 21. Oney, S., Lam, R. T., Bompiani, K. M., Blake, C. M., Quick, G., Heidel, J. D., Liu, J. Y.C., Mack, B. C., Davis, M. E., Leong, K. W., and Sullenger, B. A. (2009) Development of universal antidotes to control aptamer activity. Nature medicine 15, 1224–1228. 22. Filonov, G. S., Moon, J. D., Svensen, N., and Jaffrey, S. R. (2014) Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. Journal of the American Chemical Society 136, 16299–16308. 23. Kolpashchikov, D. M. (2005) Binary malachite green aptamer for fluorescent detection of nucleic acids. Journal of the American Chemical Society 127, 12442–12443. 24. Dolgosheina, E. V., Jeng, S. C., Panchapakesan, S. S. S., Cojocaru, R., Chen, P. S., Wilson, P. D., Hawkins, N., Wiggins, P. A., and Unrau, P. J. (2014) RNA Mango Aptamer19 ACS Paragon Plus Environment


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Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking. ACS chemical biology 9, 2412–2420. 25. Ohuchi, S., Mori, Y., and Nakamura, Y. (2012) Evolution of an inhibitory RNA aptamer against T7 RNA polymerase. FEBS open bio 2, 203–207. 26. Mori, Y., Nakamura, Y., and Ohuchi, S. (2012) Inhibitory RNA aptamer against SP6 RNA polymerase. Biochemical and Biophysical Research Communications 420, 440–443. 27. Paige, J. S., Wu, K. Y., and Jaffrey, S. R. (2011) RNA Mimics of Green Fluorescent Protein. Science 333, 642–646. 28. Pei, R., and Stojanovic, M. (2008) Study of thiazole orange in aptamer-based dyedisplacement assays. Analytical and Bioanalytical Chemistry 390, 1093–1099. 29. Sando, S., Narita, A., Hayami, M., and Aoyama, Y. (2008) Transcription monitoring using fused RNA with a dye-binding light-up aptamer as a tag: a blue fluorescent RNA. Chemical Communications 3858–3860. 30. Babendure, J. R., Adams, S. R., and Tsien, R. Y. (2003) Aptamers Switch on Fluorescence of Triphenylmethane Dyes. Journal of the American Chemical Society 125, 14716–14717. 31. Studier, F. W., and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of molecular biology 189, 113–130. 32. Butler, E. T., and Chamberlin, M. (1982) Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. Journal of Biological Chemistry 257, 5772–5778.


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33. Kim, J., Quijano, J. F., Yeung, E., and Murray, R. M. (2014) Synthetic logic circuits using RNA aptamer against T7 RNA polymerase. bioRxiv 008771. 34. Kim, J., Khetarpal, I., Sen, S., and Murray, R. M. (2014) Synthetic circuit for exact adaptation and fold-change detection. Nucleic acids research gku233. 35. Xiao, Y., Piorek, B. D., Plaxco, K. W., and Heeger, A. J. (2005) A reagentless signal-on architecture for electronic, aptamer-based sensors via target-induced strand displacement. Journal of the American Chemical Society 127, 17990–17991. 36. Rusconi, C. P., Scardino, E., Layzer, J., Pitoc, G. A., Ortel, T. L., Monroe, D., and Sullenger, B. A. (2002) RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419, 90. 37. Bompiani, K., Monroe, D., Church, F., and Sullenger, B. (2012) A high affinity, antidotecontrollable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity. Journal of Thrombosis and Haemostasis 10, 870–880. 38. Ricci, F., Vallée-Bélisle, A., and Plaxco, K. W. (2011) High-precision, in vitro validation of the sequestration mechanism for generating ultrasensitive dose-response curves in regulatory networks. PLoS computational biology 7, e1002171. 39. Cuba Samaniego, C., Giordano, G., Kim, J., Blanchini, F., and Franco, E. (2016) Molecular titration promotes oscillations and bistability in minimal network models with monomeric regulators. ACS synthetic biology 5, 321–333. 40. Blanchini, F., Samaniego, C. C., Franco, E., and Giordano, G. Design of a molecular clock with RNA-mediated regulation. 2014; pp 4611–4616. 41. Mardanlou, V., Tran, C. H., and Franco, E. Design of a molecular bistable system with



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RNA-mediated regulation. IEEE Conference on Decision and Control (CDC). 2014; pp 4605–4610. 42. Samaniego, C. C., Kitada, S., and Franco, E. Design and analysis of a synthetic aptamerbased oscillator. American Control Conference (ACC). 2015; pp 2655–2660. 43. Mardanlou, V., Samaniego, C. C., and Franco, E. A bistable biomolecular network based on monomeric inhibition reactions. IEEE Conference on Decision and Control (CDC). 2015; pp 3858–3863. 44. Cuba Samaniego, C., Giordano, G., Blanchini, F., and Franco, E. (2017) Stability analysis of an artificial biomolecular oscillator with non-cooperative regulatory interactions. Journal of biological dynamics 11, 102–120. This material is available free of charge via the Internet at http://pubs.acs.org/.


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RNA aptamer ◗ target ◗

◗ ◗

◗

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


waste

anti-sense strand (“kleptamer”)

ACS Paragon Plus Environment

reversible, cotranscriptional control of aptamer-target function


Broccoli aptamer

Kleptamer

Waste

Kb

1

A

DFHBI

A

Kb1

1

1

Full RNA kleptamer Kbf (DNA)

T

A

B

U U A C G pta U C G C me A T U r G A A A U T G G G C C G G U C U C A Kle U G G pta A me A G U C U G A G r A U G C C T C A T A C G T A C

DFHBI

A

Kb1

C

a.u.

b2 rK me

Kb2

0.5

Kleptamer added

pta

Kb2

Kbf (full kleptamer)

0.5

Kle

C C 3’ C U 5’ C C G G G G U C A

Bro

cco

li a

Broccoli aptamer

a.u.

T

HB I

DFHBI DF

0

20

Kleptamer added

40

0

Time (min)

1

20

40

Time (min)

Aptamer: 500 nM

0.9 0.8 0.7 0.6

a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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0.5 0.4 0.3 0.2 0.1 0

0

200 400 600 ACSKleptamer Paragon Plus Environment concentration

800

(nM)

1000

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DFHBI

A


Broccoli aptamer Reporter Template

Aptamer

Kleptamer

Transcription off

Waste

B

Transcription on


Inactive RNA polymerase T7 RNA polymerase: inhibition

T7 RNA polymerase: activation

1.2 1 1


0.8 0.6 0.4 0.2 0

0.80.8 0.60.6

a.u.


1

0

200

400

600

800

0.40.4 0.20.2 0 0 0 0

1000


C

SP6 RNA polymerase: inhibition

400 500

600

1000 800

1000

1500 1200

SP6 RNA polymerase: activation 11


0.8 0.8 0.6 0.6

a.u.

0.8 0.6 0.4 0.2 0

200

Kleptamer Kleptamer concentration concentration (nM) (nM)

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


0.4 0.4 0.2 0.2 00

0

200

400

600

800


ACS Environment 0 0Plus 200 500 400 600 1000800 1000 1500 1000Paragon Kleptamer Kleptamerconcentration concentration(nM) (nM)

1200


T7

A

DFHBI SP6 Aptamer

Aptamer template

B

C

SP6

T7 RNAP

Active SP6 RNAP

Inactive SP6 RNAP

250

600

Control (no aptamer template)

500

SP6 Aptamer template 120 nM Mathematical model

400

300

200

100

0

10

20

Aptamer template added

30

40

Time (min)

50

60

70

SP6 kleptamer

DFHBI

Active SP6 RNAP

Reporter

Reporter template

Waste

D

Control (no aptamer,Control no kleptamer template) SP6 Kleptamer template 1501000 nM,nM, SP6 aptamer µM Aptamer template 50 1nM

200

Aptamer nM, template nM SP6 Kleptamer template 50 1000 nM, SP6 aptamer150 1 µM Mathematical model

150

100

50

0 0

SP6

Kleptamer template Inactive SP6 RNAP

Reporter

Reporter template

T7

T7 RNAP

Reporter concentration (nM)

Reporter concentration (nM)

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0

20

Kleptamer template added

ACS Paragon Plus Environment

40

60

Time (min)

80

100

Dynamic Control of AptamerâLigand Activity Using Strand

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