Patent Grant
8110353
The current invention is directed to a synthetic catalyst based on a toehold exchange phenomenon.
Catalysts are molecules that speed up rates of target chemical reactions without being themselves consumed. Catalytic function is a necessary and ubiquitous component of life. Engineering catalysts may this allow for increased understanding of and control over biological systems. In nature, proteins are by far the most prevalent catalysts, but proteins are unfortunately difficult to engineer due to the complexity of its folding. (See, e.g., Hart W & Istrail S., Journal of Computational Biology, 4(1):1-22 (1997)) DNA, on the other hand, follows very specific Watson-Crick binding rules, and is a more suitable candidate. Additionally, many proteins denature fairly rapidly, while DNA possesses longer shelf-life. There are two basic ways of implementing DNA catalysts in the absence of proteins: to search the space of all DNA sequences to find catalytically active sequences of deoxyribozymes, and to engineer non-covalent catalysis using secondary structural properties of DNA. (See, e.g., Levy M & Ellington AD, PNAS 100(11), 6416-6421 (2003); Jaeger L, et al. PNAS, 96(26):14712-14717 (1999); and Lederman H, et al. Biochem., 45(4): 1194-1199 (2006), the disclosures of which are incorporated herein by reference.) Because it offers a more general solution (in terms of sequences), and also is more likely to function over a wider range of environmental conditions (temperature, salt, concentrations, etc.), the later is focused on in this disclosure.
In addition, nucleic acids are attractive because the combinatorial sequence space allows for an enormous diversity of signal carriers, and the predictability and specificity of Watson-Crick base pairing facilitate the design of gate architectures. The “RNA world” hypothesis further suggests that sophisticated biochemical organization can be achieved with nucleic acids alone (R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. The RNA World: The Nature of Modern RNA Suggests a Prebiotic RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 3, 2006) the disclosure of which is incorporated herein by reference), and nucleic acids have indeed been shown to be a versatile construction material for engineering molecular structures and devices (N. C. Seeman, Trends Biochem. Sci. 30, 119 (2005); and J. Bath & A. J. Turberfield, Nat. Nanotechnol. 2, 275 (2007) the disclosures of which are incorporated herein by reference), including catalytic (G. F. Joyce, Annu. Rev. Biochem. 73, 791 (2004); A. J. Turberfield et al., Phys. Rev. Lett. 90, 118102 (2003); and J. S. Bois et al., Nucleic Acids Res. 33, 4090 (2005); G. Seelig, B. Yurke, E. Winfree, J. Am. Chem. Soc. 128, 12211 (2006); and S. J. Green, D. Lubrich, A. J. Turberfield, Biophys. J. 91, 2966 (2006), the disclosures of which are incorporated herein by reference), and logical (M. N. Stojanovic, T. E. Mitchell, D. Stefanovic, J. Am. Chem. Soc. 124, 3555 (2002); M. N. Stojanovic, T. E. Mitchell, D. Stefanovic, J. Am. Chem. Soc. 124, 3555 (2002); J. Macdonald et al., Nano Lett. 6, 2598 (2006); H. Lederman, J. Macdonald, D. Stefanovic, M. N. Stojanovic, Biochemistry 45, 1194 (2006); and M. Hagiya, S. Yaegashi, K. Takahashi, in Nanotechnology: Science and Computation, J. Chen, N. Jonoska, G. Rozenberg, Eds. (Springer, New York, 2006), pp. 293-308, the disclosures of which are incorporated herein by reference) control elements and circuits (M. Levy, A. D. Ellington, Proc. Natl. Acad. Sci. U.S.A. 100, 6416 (2003); R. M. Dirks, N. A. Pierce, Proc. Natl. Acad. Sci. U.S.A. 101, 15275 (2004); M. N. Stojanovic et al., J. Am. Chem. Soc. 127, 6914 (2005); R. Penchovsky, R. R. Breaker, Nat. Biotechnol. 23, 1424 (2005); and G. Seelig, D. Soloveichik, D. Y. Zhang, E. Winfree, Science 314, 1585 (2006), the disclosures of which are incorporated herein by reference). Engineering (deoxy)ribozyme-based logic gates has been very effective, resulting in systems containing over 100 gates operating independently in parallel as well as systems demonstrating cascading of a signal between two gates. (See, Lederman H. Macdonald J, Stefanovic D, Stojanovic M N., Biochem 45(4): 1194-1199 (2006), the disclosure of which is incorporated herein by reference.) Alternatively, hybridization-based systems, usually driven by the energy of base-pair formation, have proven especially suitable for cascading signals, as demonstrated by a circuit five layers deep. (See, e.g., G. Seelig, et al., Science 314, 1585 (2006), the disclosure of which is incorporated herein by reference.) Finally, using DNA in vitro constructions of pure (non-deoxyribozyme) DNA systems also include logical circuitry (Seelig G, Soloveichik D, Zhang D Y, Winfree E.” Science 314(5808): 1585-1588 (2006), the disclosure of which is incorporated herein by reference), nanomotors and nanomachines (C. Mao, W. Sun, Z. Shen, and N. C. Seeman, Nature 297, 144-146 (1999); Yurke B, Turberfeld A J, Mills A P, Simmel F C, Neumann J L., Nature 406, 605-608 (2000); and Simmel F C and Yurke B, Appl. Phys. Lett. 80: 883-885 (2002), the disclosures of which are incorporated herein by reference), and molecular macrostructures (Goodman RP, et al., Science 310, 1661-1665 (2005); and Winfree E, et al., Nature 394, 539-544 (1998), the disclosures of which are incorporated herein by reference), as well as catalytic systems have been developed. (Turberfeld A J, et al., Phys Rev Lett 90, pp 118102.11 14; Dirks R M and Pierce N A, PNAS, 101(43): 15275-15278, 2004; and Seelig G, Yurke B, Winfree E., JACS 128(37): 12211-12220 (2006), the disclosures of which are incorporated herein by reference.)
These artificial biochemical circuits are likely to play as large a role in biological engineering as electrical circuits have played in the engineering of electromechanical devices. Toward that end, nucleic acids provide a designable substrate for the regulation of biochemical reactions. However, it has been difficult to incorporate signal amplification components.
The development of modular biochemical circuit elements poses several challenges. First, distinct signals must be carried by distinct chemical species, motivating the use of information-carrying molecules whose sequences can be used to encode signal identity. Second, “wiring up” a gate to specified inputs and outputs involves the design and synthesis of new molecules; this calls for modular gate designs. Third, a fast and robust catalytic mechanism must be identified and coupled to a suitable energy source in order to create gates with signal gain. Fourth, it must be possible to construct circuits of arbitrary complexity that can produce an unlimited variety of dynamical behaviors. Finally, there should be no leak or crosstalk between distinct signals and gates. It is difficult to meet all these challenges simultaneously. Accordingly, to date no system has been developed that would allow a rapid toehold catalysis system to be developed. Accordingly, a need exist for an improved DNA catalysis system for use in creating DNA networks.
The current invention is directed to a mechanism and system for catalyzing molecular equilibrium, using a novel catalyst design principle known as toehold exchange. In such a system, the equilibrium of a chemical reaction can be engineered to strongly favor the products by using configurational entropy as a driving force.
In one embodiment, the catalyst is cascaded into two-layer feed-forward and feedback networks, allowing access to quadratic and exponential kinetics.
In another embodiment, an allosteric version of the catalyst is presented, which can be dynamically switched between two states.
In still another embodiment, catalytic Boolean AND/OR gates are implemented and demonstrated using a variation of the catalyst design. In such an embodiment it is shown that by combining the logical AND gate with the autocatalyst, a super-exponential amplifier can be obtained.
In yet another embodiment, the catalyst system provided for use in a number of applications, such as, for example, in situ and in vivo biological detection, quantitative analysis and control.
Various examples of the present invention will be discussed with reference to the appended drawings, wherein:
a to 5e show schematics of the mechanism and test results for a exemplary toehold DNA catalyst in accordance with one embodiment of the current invention;
a to 6d show schematics of the mechanism and test results for a reporter scheme for a exemplary toehold DNA catalyst in accordance with one embodiment of the current invention;
a to 7d provide data from rate measurement experiments for the catalyst system of
a and 9b provide data from experiments on the robustness of the exemplary catalyst system of
a and 10b show schematics of the mechanism and test results for an exemplary two-layer cascaded network formed using the toehold DNA catalyst in
a to 12c show schematics of the mechanism and test results for an exemplary independent input/output catalyst system formed using the toehold DNA catalyst in
a and 13b show schematics of the mechanism and test results for an exemplary cross-catalyst circuit formed using the toehold DNA catalyst in
a to 14e show schematics of the mechanism and test results for an exemplary autocatalyst formed using the toehold DNA catalyst in
a to 15c show schematics of the mechanism and test results for an exemplary allosteric catalyst circuit formed using the toehold DNA catalyst in accordance with one embodiment of the current invention;
a and 16b show schematics of the mechanism for exemplary catalytic logic gates formed using the toehold DNA catalyst in accordance with one embodiment of the current invention;
a and 17b show schematics of the mechanism and test results for an exemplary AND gate formed using the toehold DNA catalyst of the current invention;
These drawings depict only illustrative examples of the invention and are not to be considered limiting of its scope
The current invention is directed to a catalytic design strategy that uses a novel toehold exchange mechanism that allows a specified input to catalyze the release of a specified output, which in turn can serve as a catalyst for other reactions. This reaction, which can be kinetically driven forward by the configurational entropy of the released molecule, provides an amplifying circuit element that is simple, fast, modular, composable, and robust. Using this system it has been possible to construct and characterize several circuits that amplify nucleic acid signals, including a feed-forward cascade with quadratic kinetics and a positive feedback circuit with exponential growth kinetics.
Catalytic activity has two characteristic behaviors: the speedup of the target reaction and the re-release of the catalyst to allow for multiple turnovers. To achieve these behaviors, the novel design principle of toehold exchange is used in the current invention (see,
As shown in
As an example,
Moreover, unlike previous synthetic chemical systems, the primary driving force of the catalyzed reaction can be entropy, which is a commonly-used powering mechanism of biological systems. Specifically, the reactions of the current invention may be driven using the configurational entropy gain of additional liberated molecules. For example, in regard to the schematic shown and discussed in
The fundamental reaction mechanism presented here, based on branch migration and driven by entropy, differs from the traditional view of catalysis in biological organisms in that it requires no enzymes and alters no covalent bonds. However, it is capable of molecular state changes and control, just as orthodox chemistry involving covalent bonds
It is important to ensure that alternative interactions do not interfere with intended gate functions. Toward this end, a key design principle is that the complements of the ligand domains never appear in their single-stranded form. This concept, in principle, functions for any chemistry exhibiting specific non-covalent binding between functional groups, from small polar organic molecules to polypeptides joined in quaternary structure by sterics and electrostatic.
Because the toehold catalytic mechanism of the current invention is entirely artificially engineered and a number of experimental examples using the catalyst system are provided herein. For example, using the toehold catalyst system of the current invention it is possible to cascade different systems of DNA catalysts, demonstrating the robustness of the catalyst to larger networks. It is also possible to develop catalyst networks that provide exponential kinetics by modifying the catalyst to be an autocatalyst, further showing that feedback can be achieved with catalysis. Next, an allosteric catalyst capable of dynamically switching states is demonstrated that is capable of catalyzing one of two different reactions depending on its state, thereby showing that nanomachines can serve as a control mechanism for catalysis. Finally, the current invention also demonstrates the possibility of constructing catalytic Boolean logic gates, wherein the production rate of output depends on the logical behavior of its inputs, thus integrating catalysis with logic.
As a result of the robustness and versatility of the toehold catalyst of the current invention a wide variety of potential applications are available, including, for example, in vivo detection, quantitative analysis, and control mechanisms. A number of exemplary applications, networks and circuits are described in the examples below.
The following technical specifications were used in the examples presented below.
DNA Sequence Design
The DNA sequence design process was done on a domain level. Domains denoted by unmodified numbers are termed primary domains, while domains denoted by barred numbers are termed complementary domains. First, random sequences composed of only A, C, and T were generated for each of the primary domains. Since in the intended reaction pathway only primary domains ever are exposed as single-stranded regions, this choice reduces potential secondary structure. (See, e.g., K. U. Mir, Proc. DNA Based Computers II 44, 243 (1999), the disclosure of which is incorporated herein by reference.) Sequences for complementary specificity domains were constructed accordingly. Next, subsequences known to be problematic (4 or more G's in a row on complementary domains due to G-quadruplexing, more than 4 A's in a row causing synthesis difficulties, etc.) were altered by hand. The remaining sequences were then concatenated as appropriate to form the DNA strands. These were folded alone and pairwise using the mFold web-server to determine possible spurious bindings. (See, M. Zuker, Nucleic Acids Res. 31, 3406 (2003), the disclosure of which is incorporated herein by reference.) Some bases at problematic subsequences were then changed by hand to G in the primary domains (and propagated elsewhere as appropriate for domain identity), to minimize self-folding and pairwise-folding energies. Finally, the strands were checked again on mFold to ensure minimal spurious interactions. Although only artificial nucleotide sequences are discussed above, it should be understood that the current catalyst construction mechanism is also expected to work for generalized sequences, though the kinetics of reactions involving nucleic acids with high secondary structure may be significantly slower. In a case where naturally occurring DNA and RNA sequences are used these sequences will typically incorporate all four nucleotides.
Substrate Purification
Substrate and reporter complexes were manually purified to ensure proper stochiometry and to improve purity. Sources of substrate impurity include synthesis errors and truncations, partially-formed complexes due to imperfect stochiometry, and dimerization. Strands for each sample were prepared with nominally correct stochiometry at 20 μM and annealed. For all substrate complexes except the autocatalyst substrate, the fuel strand was then added, which triggers many poorly formed substrates to decay into products that can be removed by gel purification. (For the autocatalyst, addition of the fuel strand would have initiated the exponential chain reaction, so the autocatalyst substrate was purified without addition of the fuel strand.) The samples were then run on 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) gel at 180V for 6 hours. The proper bands were cut out and eluted in TE/Mg2+ buffer for 2 days. Typical prep sizes ranged from 5 nmol to 10 nmol, and typical elution volume was 2 ml. Typical yields ranged from 40% to 60%. Purified complexes were quantified by measurement of absorbance at 260 nm, using extinction coefficients for single- and double-stranded DNA predicted by nearest-neighbor models. (See, M. Zuker, Nucleic Acids Res. 31, 3406 (2003), the disclosure of which is incorporated herein by reference.)
All annealing processes were performed with an Eppendorf Mastercycler Gradient thermocycler. The samples were brought down from 95° C. to 20° C. at a constant rate over the course of 90 minutes.
DNA Oligonucleotides
DNA oligonucleotides used in this study were purchased from Integrated DNA Technologies (IDT), with HPLC purification. Where applicable, fluorophores were attached by IDT as well.
Buffer Conditions
The buffer for all experiments was TE (10 mM Tris HCl pH balanced to 8.0, 1 mM EDTA), purchased in 100× stock from Sigma-Aldrich (catalog number T9285), with 12.5 mM MgCl2 added.
Gel Electrophoresis
Non-denaturing PAGE was run on 12% acrylamide (19:1 acrylamide:bis), diluted from 40% acrylamide stock purchased from Ambion (catalog number AM9022). ND loading dye-containing XCFF in 50% glycerol was added in 0.2× stochiometry to all samples. Gels were run at 25° C. using a Novex chamber with external temperature bath. Gels were stained with Sybr-Gold stain, purchased from Invitrogen (catalog number S-11494), and scanned with a Bio-Rad Molecular Imager. Formation gels shown in the figures were run at 180V for 1 hour.
Total RNA and Cell Lysate.
In the experiment described in
Spectrofluorimetry Studies
Spectrofluorimetry studies were done with a commercial SPEX Fluorolog-3 from Horiba. Cuvettes used were 119-004F synthetic quartz cells purchased from Hellma, with total volume 1.6 ml. For studies observing behavior of the TET fluorophore, excitation was at 524 nm, while emissions was at 541 nm. For studies observing behavior of the ROX fluorophore, excitation was at 588 nm, while emissions was at 602 nm. For studies observing behavior of the TAMRA fluorophore, excitation was at 557 nm, while emissions was at 580 nm. Slit size used was 2 nm for both excitation and emission monochrometers for net reaction studies, and 3 nm for individual rate measurements. All experiments were done with integration time of 3 seconds for every 30 second time-point. Prior to each experiment, all cuvettes were cleaned thoroughly: each cuvette was washed 15 times in distilled water, once in 70% ethanol, another 5 times in distilled water, and finally once more in 70% ethanol.
Fluorescence Normalization
All fluorescence experiments show fluorescence values normalized to approximately 1 a.u.=10 nM. Simulation traces (dotted lines) are offset vertically to correspond to quenched fluorophore baselines. Data traces within a single figure are normalized using the same scaling factor, which was determined by best-fit to simulation traces. Data traces across different figures possess different scaling factors due to differences in fluorescence reporter, lamp luminosity, and substrate concentrations. Time t=0 signals the beginning of the reaction, triggered by the addition of the last necessary reagent (usually the substrate).
Inactivity of Carrier Strands
In the course of testing the catalyst system and its derivatives, some reactions required very small quantities of certain DNA species. For example, in
An example is provided to show the basic implementation of the synthetic toehold exchange DNA catalyst. Although not required by the underlying invention, the design presented in the example shows a model catalyst system wherein a small single-stranded nucleic acid molecule catalytically releases another small single-stranded nucleic acid molecule of independent sequence from a multi-stranded complex by the process of toehold exchange. In addition, the catalyst system shows a synthetic reaction that is driven primarily by the entropy gain of molecules released, and not by the energetics of bond formation, either covalent or non-covalent.
As explained above, the toehold catalytic gate presented herein is substantially simpler than previous hybridization-based designs; moreover, it is faster, better understood, and more modular. An exemplary reaction is shown in
As previously discussed, unlike previous hybridization-based catalyst systems, the reaction design does not require unusual secondary structures such as pseudoknots and kissing loops. Moreover, undesired interactions can be avoided by design, resulting in reliable and predictable circuit behavior. (See, e.g., M. Zuker, Nucleic Acids Res. 31, 3406 (2003); J. Sager, D. Stefanovic, in DNA Computing: 11th International Workshop on DNA Computing, A. Carbone, N. A. Pierce, Eds. (Springer, Berlin, 2006), pp. 275-290; and K. U. Mir, in DNA-Based Computers II: DIMACS Workshop, L. F. Landweber, E. B. Baum, Eds. (American Mathematical Society, Providence, R.I., 1999), pp. 243-246, the disclosures of which are incorporated herein by reference.) Strands are conceptually subdivided into functional domains (number labels in
In general, toehold domains are short enough to bind only fleetingly in the absence of additional binding (and need not be distinct), but they greatly accelerate the initiation of strand displacement reactions. (See, B. Yurke, A. P. Mills, Genet. Program. Evolvable Mach. 4, 111 (2003), the disclosure of which is incorporated herein by reference.) Specificity domains, meanwhile, ensure specific interactions [even a single mismatch can slow down branch migration substantially (I. G. Panyutin, P. Hsieh, J. Mol. Biol. 230, 413 (1993), the disclosure of which is incorporated herein by reference) and determine the identities of the catalyst and output molecules. The lengths of the toehold domains determine kinetics and need to be between roughly 4 and 10 nucleotides (nt), but the specificity domains may be of any length sufficient to ensure thermal stability. Domains 1 and 6 of OB and SB, respectively, are inert, whereas their respective toeholds are sequestered in S.
In the toehold exchange reaction of the current example, as shown in
In
The kinetics of the model system were monitored using independent reporter complexes OR and SR. This approach was chosen (rather than direct labeling of strands in the catalyst system) to decouple the thermodynamic effects of fluorophore-quencher binding from the catalytic pathway. Both OR and SR initially contain a 20 bp duplex and a 7 nt toehold domain that uniquely binds their respective targets (OB and SB). Each possesses a different fluorophore and quencher pair (TET and Iowa Black Fluorescence Quencher (FQ) for SR; ROX and Iowa Black Red Quencher (RQ) for OR). The reactions are assumed to be non-reversible, as given by the reaction schematics below:
Displacement rate constants of the two reporter complexes OR and SR were measured via fluorescence at initial reactant concentrations of 1 nM ( 1/30 the concentration used in catalyst experiments) to be kTET=8·105 M−1s−1 and kROX=4·105 M−1s−1, as shown in
During the operation of these reporter, OR reacts stochiometrically with output OB to separate a fluorophore-labeled strand from a quencher-labeled strand, thereby increasing fluorescence (
This system was modeled using the reduced reaction set shown in the reaction schematic below.
where k0=2.3·101 M−1s−1, k1=6.5·105 M−1s−1, k2=4.2·105 M−1s−1, k3=4·10−3 s−1 (fitted), and kROX=4·105 M−1s−1. The first reaction shown models an uncatalyzed (leak) reaction. Intermediate steps in branch-migration reactions are omitted, because they are relatively fast at experimental concentrations (C. Green, C. Tibbetts, Nucleic Acids Res. 9, 1905 (1981), the disclosure of which is incorporated herein by reference) and because intermediates I1, I2, and I4 are not observed in PAGE analysis of reactants and products (
Using kTET and kROX rate constants from the previous section, reaction rate constants k1, k2, and k0 are directly measured to be 6.5·105, 4.2·105, and 2.3·101 M−1s−1, respectively, as shown in
Note that for these experiments, the k−1 rate of reverse reaction SB+I3 can effectively be ignored, because SB was consumed by reporter complex SR (simulations showed no visible difference when k−1 was modeled). The last rate constant k3 is difficult to measure because it is first-order, and the rate could not be slowed down to a time-scale where spectrofluorimeter readings would be meaningful. Thus, for the purposes of this analysis it was fitted using the results of the net kinetics of the catalytic system to be 4·10−3 s−1. Using this analytic framework the time course of the catalyzed reaction over a wide range of catalyst concentrations is accurately reproduced by this reduced system of rate equations (
As shown in the reaction schematics provided in
To understand this result, consider the net reaction:
S+FOB+SB+W
The free energy change for this reaction, in dilute solutions, is given by:
ΔG=ΔGOB°+ΔGSB°+ΔGW°−ΔGS°−ΔGF°+RT ln Qdef=ΔGnet°+RT ln Q
where Q=([OB]/c°·[SB]/c°·[W]/c°)/([S]/c°·[F]/c°) is the reaction quotient relative to standard conditions and ΔG°x is the standard free energy of species X at standard conditions, which here specify the TE buffer with 12.5 mM magnesium, 25° C., and c°=1 M.
The free energy change (the driving force for the reaction) decreases as concentrations change during the course of the reaction; once equilibrium is achieved, Q=exp{−ΔG°net/RT} and ΔG=0. If the standard free energy change ΔG°net≈0, as would be expected for the reaction with the full-length fuel strand if the standard free energy is dominated by base pairing, then the driving force at any moment is just RT In Q. As a somewhat arbitrary reference point, the time at which half the substrate has been depleted can be considered. For the reaction in
The free energy difference between the substrate S and the maximally truncated waste product W was approximated using the mFold server using DNA parameters for 25° C., with salt conditions being 10 mM Na+ and 12.5 mM Mg2+. Taking into consideration the 8 base pair stacks, external loops and dangles (due to the 1 domain in S, and the 3′ overhang on the LB strand on the truncated waste product W), and an initiation entropy of 6.4 cal/mol/K per association, the predicted standard free energy change ΔG°net for the (unfavorable) forward reaction is +11.7 kcal/mol.
According to these estimates, truncating the fuel strand F by 8 bases should disfavor the forward reaction enough that the equilibrium distribution possesses substrate S in excess of waste W. To verify the entropic driving force analysis was conducted by PAGE (12% native gel) of reactions with truncated fuel strands. In this experiment [S]=[F]=200 nM, [C]=20 nM, as denoted by the asterisk. All reactions were run at 25° C. for 3 hours. “Ft2” denotes that two bases were truncated from the 5′ end of fuel strand F. However, the experiments described in
The thermodynamic driving force of the exemplary toehold catalyst, being dominated by center-of-mass configurational entropy of released molecules, is expected to be robust to environmental conditions such as temperature and salt concentrations that alter the strength of DNA hybridization. Specifically, salt conditions affect the free energy of hybridization (J. Santa Lucia, Proc Natl Acad Sci USA 95, 1460 (1998), the disclosure of which is incorporated herein by reference), but since there is no net gain or loss of base-pairs in a net reaction cycle, the equilibrium should not be significantly affected. The strength of the binding of the toehold domains still depend on the free energy of the base pairs formed, so salt concentration will affect the kinetics of the catalyzed pathway. However, the catalyst should qualitatively function across the range of salt concentrations, as long as the toehold domains still are able to co-localize the relevant strands. In
Meanwhile, temperature affects the equilibrium of a reaction only through the enthalpic change (ΔH°). When the magnitude of the enthalpic change is small (ΔH°≈0) as it is for the entropy-driven system, the effects of temperature have little effect on the reaction equilibrium although, again, they affect the kinetics of toehold-mediated processes. In
Finally, in addition to the relative insensitivity to the catalyst system of the current invention to environmental conditions such as salt concentration and temperature, it has also been demonstrated that the function of the DNA catalysts is relatively independent of substrate concentration. In exemplary studies the reaction was operational in substrate concentrations of from 1 nM to 100 nM, and theoretical analysis indicates that such reactions should operate in substrate concentrations of from about 1 pM and 10 μM.
In order for an engineered catalyst system to be integrated into large circuits for complex dynamic behavior, it is essential that several instances of the catalyst system can be cascaded. As a first proof-of-principle example, two two-layer feed-forward circuits are constructed by designing second catalyst systems with outputs containing a subsequence that acts as the catalyst for the original systems described in Examples 1a and 1b above. Specifically,
As shown in
The concentration of upstream catalyst C0 is constant, so initially [OB0] increases linearly with time, which causes [OB1] to increase quadratically with time (
As mentioned, the downstream layer catalyst system of
This cascaded system can also be used as an amplifier to detect small quantities of C0. Repeated fluorescence experiments show that it is possible to distinguish reliably between 1 pM (0.0001×) catalyst C0 and 0× catalyst within 12 hours (
Several factors can reduce the repeatability of fluorescence experiments: First, the spectrofluorimeter luminosity output differs from lamp bulb to lamp bulb and luminosity tends to decrease as any particular lamp bulb ages. Second, different preparations of purified substrate complexes S, though nominally calibrated to the same concentration, in practice differed in purity. Third, fluorophores tend to bleach, and thus older stocks tend to give lower fluorescence readings for the same concentration. Finally, the Eppendorf pippetors used in these studies are high precision but low accuracy; thus using two different pipetors to measure the same volume would often yield different pipetted quantities.
To minimize these effects the following steps were taken for all fluorescence experiments displayed:
The domains involved in the quadratic feed-forward circuit shown in
This catalyst system functions almost identically to the one presented in
Finally, feedback in this two-layer circuit can be achieved by redesigning OB1 so that it can, in turn, catalyze the F0+S0 reaction. A schematic of such a circuit is shown in
Feedback in this cross-catalytic system causes the concentrations of both OB0 and OB1 to grow exponentially at early times, as is shown in kinetic data from fluorescence studies provided in
As illustrated above, the only difference between the cross-catalytic circuit and the feed-forward circuit is the identity of the 5′ domain of the OB1 strand; this difference should not affect any rate constants, so the rate constants used in simulations are exactly the same as those used for the quadratic feed-forward circuit. The only additional parameter that needs to be fit is the effective initial concentrations of OB0 and OB1E.
Although embodiments of “feedback” networks capable of producing exponential kinetics are provided above, such kinetics can also be achieved with a much smaller autocatalytic system by modifying the substrates S of the catalyst systems presented in
In one exemplary embodiment, exponential growth kinetics are achieved by redesigning the reaction presented in
d shows that the exemplary autocatalytic system has this characteristic behavior, implying that exponential growth kinetics have indeed been achieved and that the reaction is not substantially affected by product inhibition. Further confirmation comes from the quality of fit to the data of a model based on rate constants derived for the catalyst system of
Parameters kTET, k0, k1, k−1, k2, and k−3 again are the same as measured previously. Parameter k3=4·10−3 s−1 is the same as fitted to the catalyst data in
Largely because of their relevance to the origin of life and to the RNA world, autocatalytic and cross-catalytic self-replication reactions have been proposed and demonstrated previously. (See, e.g., R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. The RNA World: The Nature of Modern RNA Suggests a Prebiotic RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 3, 2006); and N. Paul, G. F. Joyce, Curr. Opin. Chem. Biol. 8, 634 (2004), the disclosures of which are incorporated herein by reference.) However, such systems typically suffer from product inhibition and thus exhibit parabolic, rather than exponential, growth kinetics. Recent exceptions include cross-catalytic deoxyribozymogens (M. Levy, A. D. Ellington, Proc. Natl. Acad. Sci. U.S.A. 100, 6416 (2003), the disclosure of which is incorporated herein by reference) and catalyzed self-assembly (P. Yin, H. M. T. Choi, C. R. Calvert, N. Pierce, Nature, in press, the disclosure of which is incorporated herein by reference) based on the hybridization chain reaction (R. M. Dirks, N. A. Pierce, Proc. Natl. Acad. Sci. U.S.A. 101, 15275 (2004), the disclosure of which is incorporated herein by reference); as described herein the current autocatalyst system is substantially faster than these prior art systems. This increase speed is the result of the reduced spontaneous activity of the circuit (for example, by improved purification of the substrate complex), and is important to ensure that the system can be used as an enzyme-free constant-temperature alternative to PCR for detecting known sequences.
For many applications in biotechnology, nucleic acid devices must remain functional in the presence of naturally occurring macromolecules. The autocatalyst system of the current invention was therefore tested in the presence of an excess of mouse liver total RNA with rabbit reticulocyte lysate (
To establish greater control over kinetics of catalysis, it is possible to model the action of DNA nanomotors. In those constructions, a DNA molecule is able to switch between several different states upon the exogeneous addition of actuator strands. This idea can be incorporated into the catalyst system of the current invention in the form of an allosteric catalyst that adopts one of two hairpin configurations (see
Building upon the work of the allosteric catalyst presented in
In previous work in vitro logic gates have been constructed from DNA complexes based on hybridization kinetics. These gates, however, are stochiometrically consumptive, and are difficult to integrate into complex networks without robust signal restoration. In the current example designs are presented for catalytic logic gates using the entropy drive DNA catalyst system, such that the inputs act as catalysts and are not consumed.
The ability to construct larger circuits will enable the wide range of chemical circuit functions needed for sophisticated applications. The current toehold exchange catalytic reaction networks are suited for scaling up to larger circuits. The modular molecular design makes synthesis of more complex components and networks with arbitrary topology straightforward. To demonstrate this, schematics for logical AND and OR gates are presented in
A functional embodiment of an AND gate and experimental results for the logical AND gate function are shown in
The multiplicative (AND-like) behavior can be understood quantitatively as follows: the left (5′) and right (3′) catalytic ends operate independently and follow approximately the same kinetics as the catalyst system of
As the 3′ region of the output and the substrate are very similar to the system given in
By combining the ideas of the autocatalyst and the logical AND gate, a simple circuit can be formed that is capable of displaying super-exponential kinetics (see
As shown in the above examples, the novel toehold exchange mechanism used in the catalyst system of the current invention allows a specified input to catalyze the release of a specified output, which in turn can serve as a catalyst for other reactions is provided. This reaction, which can be driven forward by the configurational entropy of the released molecule, provides an amplifying circuit element that is simple, fast, modular, composable, and robust. Using this system it has been possible to construct and characterize several circuits that amplify nucleic acid signals, including a feed-forward cascade with quadratic kinetics and a positive feedback circuit with exponential growth kinetics. Moreover, the system is extremely sensitive. For example, using the feed-forward circuit, 1 pM of DNA can be specifically detected in the course of 12 hours. A minimal autocatalyst is designed as a variant of the catalyst that also exhibits exponential growth. Finally, an allosteric catalyst that can be dynamically switched, and a sigmoidal activation function were also experimentally demonstrated. Because of the flexibility of the catalytic system of the current invention is the availability of numerous potential applications.
One application of being able to engineer an arbitrary sequence into a catalyst is real-time minimally interfering measurement of the concentration of the molecule. For example, in a cell any particular mRNA sequence is constantly being produced and degraded, and the dynamics of its concentration may be of interest. Furthermore, we would not wish to deplete the mRNA in the process of quantitation (e.g. titration versus its complement), as the sequence of interest may be essential to the cell's function/survival. Quantitative PCR (qPCR) has been long used to detect and quantitate specific sequences of DNA and RNA in vitro, but qPCR requires both biologically farmed enzymes and temperature cycling for proper function, thus its application for in vivo systems is not feasible. The quadratic feed-forward circuit provides an intermediate in the tradeoff between operation time and detection sensitivity, and has been experimentally demonstrated to be able to detect 1 pM in the course of 12 hours. A single molecule in a eukaryotic cell volume also corresponds to about 1 pM. Thus, feed-forward circuits with polynomial kinetics may be a reasonable alternative for DNA detection and quantitation.
For scaling up to large circuits, independent catalyst systems must have negligible crosstalk. The success of quantitative models that assume no crosstalk, as presented above, is encouraging; further evidence comes from a test of two independent catalyst systems operating in the same solution. Experimental data from this test is provided in
In short, the catalytic systems of the current invention have the potential to avoid the slowdown that plagued previous attempts to construct large nucleic acid circuits. Future nucleic acid control circuits can be interfaced to molecular sensors and actuators. This may be achieved directly when the inputs and outputs are themselves nucleic acids, such as for the detection, analysis, and response to complex nucleic acid samples (Y. Benenson, B. Gil, U. Ben-Dor, R. Adar, E. Shapiro, Nature 429, 423 (2004), the disclosure of which is incorporated herein by reference), or for the control of nucleic acid nanomachines (R. Pei et al., J. Am. Chem. Soc. 128, 12693 (2006), the disclosure of which is incorporated herein by reference). Nucleic acid circuits can also respond to and control more general chemical events: in principle, the release of an oligonucleotide could regulate covalent chemistry by controlling (deoxy)ribozyme activity (9) or reactant proximity. (See, X. Li, D. R. Liu, Angew. Chem. Int. Ed. 43, 4848 (2004), the disclosure of which is incorporated herein by reference.) Additionally, signals carried by small organics and other non-nucleic acid molecules can be read by nucleic acid systems with the use of aptamer domains (A. D. Ellington, J. Szostak, Nature 346, 818 (1990); and J. Tang, R. R. Breaker, Chem. Biol. 4, 453 (1997), the disclosure of which is incorporated herein by reference), and other binding interactions that can regulate toehold accessibility (S. Müller, D. Strohbach, J. Wolf, Proc. IEEE Nanobiotechnol. 153, 31 (2006); and F. J. Isaacs, D. J. Dwyer, J. J. Collins, Nat. Biotechnol. 24, 545 (2006), the disclosure of which is incorporated herein by reference). Thus, nucleic acids could provide a general-purpose system for the synthesis of embedded control circuitry within aqueous chemical systems.
Although examples of a toehold exchange catalyst system and its use have been described and illustrated in detail, it is to be understood that the same is intended by way of illustration and example only and is not to be taken by way of limitation. For example, the invention has been illustrated in the form of a DNA catalyst using specific sequences. However, the toehold exchange catalyst system may be configured with other kinds of sequences or using other kinds of ligands including non-nucleic molecules. Accordingly, variations in and modifications to the toehold exchange catalyst system and its use will be apparent to those of ordinary skill in the art. In most cases, and as will be readily understood by one skilled in the art, alternative configurations of the system may be substituted with small changes, such as, for example, lengths of domains, 5′/3′ orientation of molecules, RNA or PNA analogs, etc. Furthermore, throughout the exemplary embodiments, where components are illustrated, these may be substituted as is known in the art within the scope of the invention. The following claims are intended to cover all such modifications and equivalents.
The current application claims priority to U.S. Provisional Application No. 60/899,546, filed Feb. 5, 2007, the disclosure of which is incorporated herein by reference.
The U.S. Government has certain rights in this invention pursuant to Grant No. DMS-0506468 awarded by the National Science Foundation.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6022714 | Brown et al. | Feb 2000 | A |
| 6642014 | Pedersen et al. | Nov 2003 | B1 |
| 6696285 | Mills, Jr. et al. | Feb 2004 | B1 |
| 20040062808 | Langrana et al. | Apr 2004 | A1 |
| 20070072215 | Seelig et al. | Mar 2007 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20090191546 A1 | Jul 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60899546 | Feb 2007 | US |
