Patent Grant
12054713
Producing nano-scale computers with molecules offers substantial potential, in part because such computers may be well-suited for solving certain computation problems. In particular, computers employing biomolecules can be compatible with biological environments, rendering them amenable for use in complex-disease diagnostics or even treatments.
The ability to translate one nucleic acid sequence into another in principle allows one to build logic gates and networks with nucleic acids. These gates and networks are driven by two events, hybridization and strand displacement, both of which generally are thermodynamically favorable, i.e., they involve a transition from a higher—to a lower-energy state. Thus, both events can occur spontaneously in a system.
Hybridization involves free, single-stranded stretches of nucleic acids. Accordingly, a nucleic-acid network may be regulated by the availability of these free strands.
A “sequestering event” allows certain sequences to be available conditionally to the rest of the network. Such events empower the construction of translators, which convert one single-stranded nucleic acid sequence into a different single-stranded nucleic acid sequence. These translators are the foundation on which can be built, with nucleic acids, basic logic operators such as AND, NOT, OR, NAND, NOR, XOR and XNOR. From these and other logic components, larger networks can be constructed that include components such as amplifiers. Accordingly, these translation events are important for information processing with nucleic acids and molecular computing.
In accordance with one aspect of the present invention, provided is a composition comprising a first and a second nucleic acid complexes each comprising a first, a second, a third, and a fourth nucleic acid strands, each of the strands comprising, sequentially, a first, a second and a third fragments, wherein the nucleic strands are defined as B-X-D,
In one aspect, the composition further comprises a third nucleic acid complex comprising a first and a second nucleic acid strands, each of the strands comprising, sequentially, a first, a second and a third fragments, wherein the first and second strands are defined as E-Z-H and
the third strand of the second complex (F-Z-G) has suitable sequence complementarity to the second strand of the third complex (
In some aspects, the third fragment (Ē) of the third strand of the first complex and the first fragment (E) of the first strand of the third complex have sequence complementarity but do not stably bind to each other under normal hybridizing conditions due to chemical modification to either or both of the fragments; and the first fragment (F) of the fourth strand of the first complex and the third fragment (
Chemical modifications suitable for practicing the present technology include, without limitation, replacing sugar-phosphodiester backbone of a nucleic acid fragment with a mini-PEG-conjugated, serine-derived gamma-PNA. In some aspects, the chemical modification comprises substituting a tricyclic cytosine analogue for the nitrogenous base of at least one nucleoside in a nucleic acid fragment. In one aspect, the chemical modification comprises introducing a heteroatom at the 2′-position of a sugar moiety in a nucleotide.
Illustrations of the complexes and their relationships can be found in
In one aspect, each fragment is from about 5 bases to about 50 bases long. In another aspect, each each fragment that is single-stranded is from about 5 bases to about 30 bases long.
It also is within the scope of the disclosure that each composition can further optionally comprise a pharmaceutically acceptable carrier. Some embodiments of the present disclosure, further, provide a cell comprising the composition or complex of the present disclosure.
Computer-implemented methods and non-transitory computer-readable media suitable for simulating, designing, recording, reporting, or analyzing certain aspects of the disclosed technology are also provided here.
Provided as embodiments of this disclosure are drawings which illustrate by exemplification only, and not limitation, wherein:
Some or all of the figures are schematic representations for exemplification; hence, they do not necessarily depict the actual relative sizes or locations of the elements shown. The figures are presented for the purpose of illustrating one or more embodiments with the explicit understanding that they will not be used to limit the scope or the meaning of the claims that follow below.
Solid-phase sequestering, toe-hold sequestering, and toe-hold exchange are among the various approaches that can be used in translating nucleic acid sequences to build logic operators and networks. Described in greater detail below, these three particular approaches are exemplified via geometries that utilize three-way, toe-hold-mediated branch migration reactions. Additional mechanisms are possible for branch migration reactions, including but not limited to four-way branch migration, four-way accelerated migration, and multi-strand complex migration.
Thus, while there are embodiments described below that employ three-way branch migration for illustration purposes, the present invention contemplates DNA logic gates and networks built to utilize other branch migration pathways. Conversely, the embodiments of the invention can be applied to any branch migration reaction.
Throughout this disclosure and in the accompanying figures, capital letters, e.g., A, B, C, X, Y, Z, optionally with subscripts or superscripts, are used to represent a stretch, also referred as a “fragment,” of oligonucleotides of arbitrary length. The corresponding A′, B′, C′, X′, Y′, Z′ or interchangeably Ā,
The terms “oligonucleotides,” “polynucleotides” and “nucleic acids” are used here to encompass all forms of nucleic acid molecules. Without limitation, this category includes ribonucleic acids (RNA), deoxyribonucleic acid (DNA), peptide nucleic acids (PNA), and their derivatives, with and without modifications, respectively.
Solid-phase sequestering entails physical separation of the relevant sequences/strands in space by means of beads, nanoparticles, or surfaces. This approach employs principles of site isolation, which have found extensive use in organic chemistry. The timing of these displacement events in solid-phase sequestered geometries can be controlled by regulating whether the necessary strands are in the solution phase or in the solid phase of the system.
The strands bound to one solid surface interact extremely slowly with strands on another solid surface due to steric effects. Consequently, the strands in the solution phase are the only components that can interact with the solid phase operators.
Toe-hold sequestering accomplishes the same operations as solid-phase translators, but functions by keeping stretches of sequence bound up in a duplex. Just as in the solid-phase translator, a displacement event can free the sequence of interest. All of the strands can be in solution together, by the consequence of which displacement events are regulated by the availability of toe-holds. The term “toe-hold” here refers to short stretches of single-strand nucleic acid sequences that provide a starting point for a displacement event.
While potentially very useful, applications of the toe-hold geometries have been limited to date by the rate at which a system containing such toe-hold geometries can propagate information. Inherent limitations in conventional toe-hold-sequestered approaches frequently can slow the resultant propagation down below a biologically useful timescale. More specifically, toe-hold sequestered translators work at reasonable speeds only in a narrow dynamic range of concentrations as a consequence of the clashing interactions. By tuning the length of toe-holds, clashing interactions can be made reversible at lower concentrations. As concentration increases, however, clashing would dominate, and the system would grind to a halt. If concentration decreases, on the other hand, toe-hold mediated displacement would also grind to a halt. In this context, it is noted that, in a biological context, concentrations can vary widely; hence such translators are poorly suited for use in the actual biological context. It is believed that kinetic bottlenecks are a result of unproductive reactions, referred to here as “toe-hold clashes,” that occur when a toe-hold is bound by a molecule having a complementary sequence or “clashing strand” that cannot produce a displacement reaction.
For instance,
Another limitation of this approach is the potential toxicity resulting from exposure of the the large single-stranded regions of the translators. For instance, when a single-stranded region binds to endogenous DNA or RNA in a cell, it can alter transcription, translation, or other function of the DNA or RNA molecule. Such “off-target” event can lead to undesired consequences in the cell.
Yet another limitation is the potential signal leak due to background reactions of translators in the absence of exposed toe-holds, which can also lead to undesired consequences. For instance,
One proposed solution is to keep toe-holds short to mitigate the effect of clashes on the system: the shorter the toe-hold is, the faster the on/off rate of the complementary sequence can be. Thus, five or six nucleotide-long toe-holds are common because at these lengths, if a non-productive binding event occurs, the time spent in the double-stranded, “clashed” state is short.
This approach creates the aforementioned kinetic bottleneck, however, because the productive binding event is constrained by the same thermodynamic parameters; hence, the incoming strand likewise does not bind strongly to these toe-holds. Consequently, the desired displacement does not always occur when the correct incoming strand binds, as it needs to be in the bound state long enough to initiate the displacement reaction. The use of short toe-holds thus increases the amount of time required for a given operation to occur and produce an output. Put another way, the displacement reaction cannot take place before the occurrence of many binding events, both by clashing strands and by desired strands. This inefficiency limits the utility of the system by slowing down the propagation of information to time scales that are too extended to be useful.
Shorter toe-holds, along with shorter single-stranded regions on the translators, can also help reduce toxicities. However, the shorter toe-holds do not solve the problem with signal leak.
“Toe-hold exchange,” uses similar pairing interactions as toe-hold sequestering but with different geometrics. Toe-hold exchange attempted to solve the toe-hold clash problem posed by the toe-hold sequestering approach. With reference to
The toe-hold exchange approach has inherent limitations too, however. First, like in toe-hold sequestering, the long single-stranded regions of the translators can cause undesired toxic effect due to off-target binding to endogenous nucleic acids in a cell.
Another issue relates to the dynamic range of the translation which is similar to, albeit not the same as, the issues encountered in toe-hold clashing. This issue is less severe with toe-hold exchange, though, since it impacts speed of translation more so than yield. In this case the lengths of the toe-holds determine the rate of spontaneous dissociation, which is faster with shorter toe-holds. On the other hand, they also determine the forward displacement rate, but longer toe-holds make forward displacement faster. Therefore, there is a balance between these constraints in a concentration dependent way.
An important limitation with the toe-hold exchange approach is the low yield, as all reactions are bi-directional, leading to no favoring of final product unless, for instance, the entire translation is coupled to an irreversible reaction at the end.
The present disclosure provides a series of translators that solve the problems posed by the approaches detailed above. With these translators, toxic off-target effects and background reactivity are minimized, toe-hold clashes can be avoided, the overall yield is close to complete, and the translation speed still is improved significantly.
The second nucleic acid complex (102) shown in
By virtue of sequence complementarity in the hanging single-stranded regions, the first complex (101) has suitable sequence complementarity so as to be capable of binding to the second complex (102), which initiates a strand displacement reaction that involves six strands. The output of the strand displacement reaction includes two completely annealed duplexes (105 and 106) and a new dual toe-hold (107), having a duplex region in the middle and four separate single-stranded regions (
The newly formed dual toe-hold, comprised of
As explained above, the strand displacement reactions in this approach achieve high yield rate because each of the reaction generates output products at a much lower energy state than the input nucleic acid molecules. As Table 1 illustrates, moreover, experimental data show that such dual toe-hold-mediated reactions can be quite fast as well. Compare the column of “Dual Toe-Holds” to the ones identified as “3′ Toe-hold” or “5′ Toe-hold”.
In accordance with these findings, the present disclosure provides a number of translators and translator sets, sometimes referred to as “adaptors.” A translator or translator set takes a nucleic acid molecule (e.g., single stranded nucleic acid, complete or partially duplex nucleic acids, or more complex nucleic acid structure or translators) as input and carries out one or more strand displacement reactions to produce one or more nucleic acid molecules as outputs. Each of these inputs or outputs can serve as outputs or inputs to other translators or translator sets in a system. Each of these translators and translator sets therefore functions as a logic operator and, in combination, carries out desired computation and information conveyance.
For instance, in
Accordingly, in one embodiment the present disclosure provides a composition comprising first and second nucleic acid complexes, each comprising first, second, third, and fourth nucleic acid strands. Each of the strands comprises sequentially (i.e., from 5′ to 3′, consecutively or separated with intermediate nucleotides) a first fragment, a second fragment and a third fragments, where the nucleic strands are defined as:
In one aspect, each of the first and the second complexes comprises a first duplex region formed between the second fragments of the first and second strands (X::
In each of the first and second complexes, the first fragment (B and
Further, the third strand of the first complex (
In one aspect, the composition further comprises a third nucleic acid complex comprising a first and a second nucleic acid strands, each of the strands comprising, sequentially, a first, a second and a third fragments, wherein the first and second strands are defined as E-Z-H and
The present disclosure also provides a methodology for preventing toe-hold clashing. As shown in
In one such method, each nucleic acid fragment that can potentially be exposed as single-stranded during the translation process is subject to potentially chemical modification. In addition to its natural state (state N, annotated as subscript in, e.g., CN), each of such fragment can be modified to form two states, states R (e.g., right-handed) and L (left-handed), respectively, so long as such modifications allow (or promote) and prohibit (or inhibit) binding between complement strands of certain states as shown in Table 2 below.
With such modifications, in one aspect the tail fragment (ĒL) of the third strand of the first complex (102) has reverse sequence complementarity with the head fragment (ER) of the first fragment of the first strand of the third complex (104), but does not stably bind to it. Likewise, in another aspect the head fragment (FL) of the fourth strand of the first complex (102) has reverse sequence complementarity with the tail fragment (
As used herein, the term “does not stably bind” indicates that two nucleic acid strands do not form a stable duplex structure, even if they share sequence complementarity. A “stable” duplex structure, in some aspects, refers to a duplex that can exist in a non-transient fashion at a concentration that is higher than 0.1%, or 1%, or 5%, or 10% of the total concentration of each individual strand in a reaction solution.
As further described below, chemical modifications are known that can achieve the binding objectives defined in Table 2. “Chemical modification” in this context is not limited to chemically modifying a nucleic acid molecule after it is produced; rather, the phrase also encompasses incorporating chemically modified nucleotides while producing the nucleic acid molecule.
There are many well-characterized nucleic acid modifications that can be used to modulate the thermodynamic properties of binding to natural DNA or RNA. These include changes to the backbone, sugar, or nucleobase of the oligonucleotide. The modifications also can be employed separately or in conjunction with one another; that is, using a modified backbone does not preclude the use of a modified nucleobase in the same strand.
Nucleic acid backbone analogs can be used to improve the binding of strands capable of producing a displacement reaction. There are a number of different analogs that could be used, all of which offer tighter binding affinities to DNA and RNA than natural nucleic acids. These analogs include but are not limited to those with uncharged backbones (peptide nucleic acids or phosphorodiamidates), positively charged backbones (guanidinium peptide nucleic acids), and hydrogen-bonding groups that allow for pre-organization (gamma peptide nucleic acids). In one aspect, the backbones include mini-PEG-conjugated, serine-derived gamma-PNAs, as illustrated in
General structures for certain analogs are shown in
The use of modified sugar rings can also alter the thermodynamics of binding to DNA or RNA for an oligonucleotide. The most widely used analogs are morpholinos, locked nucleic acids (LNAs), and LNA derivatives. Other modified sugars are documented in the literature that also could produce a similar result, in terms of altering binding thermodynamics. Illustrative of these are sugars with modifications at the 1′, 2′, 3′ or 4′ position and sugars with different atoms substituted for the oxygen in the ribose cyclopentane ring. These analogs are illustrated in
Nucleobase modifications can also be used to achieve the same effect as backbone and sugar analogs; namely, altering the thermodynamics of specific hybridization reactions. These bases include methylcytosine, diaminopurine, G-clamp, and phenoxazine (
Another chemical modification that could be used to alter the thermodynamics of binding interactions is the incorporation of charged polymers like chitosan, which has been shown in the literature to accelerate the rate of displacement reactions. However, since these polymers speed up reactions non-specifically, they would have to be used in conjugation with one of the other modifications mentioned above to allow for discrimination between desired and undesired binding events.
Different approaches can be used to synthesize the molecules with the chemical modifications discussed above. For example, the backbone chemistry can be taken into consideration of the design of the modified molecules. Backbone chemistry is what is used to put together individual monomers into a longer strand. Modifications that involve the nucleobase or the sugar but that keep the natural phosphodiester backbone of DNA/RNA can be synthesized via standard phosphoramidite chemistry, as employed for natural monomers. Illustrations of these methods are found, for example, in Beaucage, S., and R. Iyer, Tetrahedron 48: 2223 (1992), in Brown, D. M. A, “Brief history of oligonucleotide synthesis,” 20 M
If the backbone is changed in a particular modification, a different chemistry will be employed. Such modification chemistry is described in the scientific literature. Thus, peptide nucleic acids (PNAs) and their derivatives rely on amide bonds to link the individual monomers together. Instead of using phosphoramidite chemistry, therefore, strands of these monomers are made with amide bonding forming conditions and coupling reagents like HBTU. An exploration of the methods used to make PNA or PNA-like oligonucleotides can be found, for instance, in F. Beck, “Solid Phase Synthesis of PNA Oligomers,” M
These chemical modifications include introduction of handedness into a DNA or PNA fragment. Handedness of DNA, particularly of PNA, is a well-characterized feature, and methodology for generating handedness also is known. See, e.g., Corradini et al. “Control of helical handedness in DNA and PNA nanostructures,” M
Another backbone modification approach involves chimeric oligonucleotides. These are oligonucleotide strands that contain different backbone chemistries in the same molecule. For example, if one needed a strand that was half PNA backbone and half DNA backbone, one would need a way to join these two different backbone chemistries. Making these chimeric strands is also generally known in the art. In the above example of a PNA/DNA chimera, the difference in chemistries can be bridged by using modified DNA or PNA monomers. For DNA, the 5′-dimethoxytrityl (DMT) protected hydroxyl is replaced with a monomethoxytrityl (MMT)-protected amine that can react with the carboxylic acid of a PNA after deprotection. For PNA, the protected N-terminal nitrogen is replaced with a DMT-protected hydroxyl that can react with the phosphoramidite group on DNA after de-protection. These approaches are further described, for instgance, in E. Uhlmann et al., Angew. Chem. (Int'l ed.) 37: 2796-823 (1998).
All of these modifications, whether used individually or in conjunction with one another, can affect the thermodynamic conditions of specific interactions in an arbitrary nucleic acid network such that the binding of desired strands or complexes is favored over clashing interactions without altering sequence content. All of these interactions can apply to any branch-mediated migration reaction, whether they are 3-way branch migrations, such as solid-phase sequestering, toe-hold sequestering, or toe-hold exchange, or are branch migrations that take place by other mechanisms, e.g., four-way branch migration, four-way accelerated branch migration, or multi-strand complex migration.
Translators provided in this disclosure can be used to build nucleic acid computers, which can be useful medically. For instance, the translators can “sense” the presence of a viral nucleic acid and then carry out a series of translation events, resulting in the “release” of a regulatory RNA (e.g., siRNA, antisense RNA) that can be used to initiate an antiviral response or apoptosis of the host cell, in order to eliminate the virus.
I. mRNA Adaptor
“Sensing” can be implemented as initiation of one or more strand displacement reactions, taking a nucleic acid (e.g., viral DNA/RNA, tumor DNA/RNA) as input, and releasing one or more toe-holds as output. The translators that carry out such one or more strand displacement reactions are termed “adaptors.” In the case of a sensing a tumor mRNA, for instance, such an adaptor can be called a “mRNA adaptor.”
An mRNA adaptor is illustrated in
The second complex in the mRNA adaptor (203) includes at least three strands. A first strand (P-B-Ā) is partially complementary to the second strand of complex 202, forming two separate duplex regions (Ā::A and B::
In a cell that contains the mRNA adaptor that includes complexes 202 and 203, therefore, upon presence of mRNA molecule 201 the mRNA initiates a strand displacement reaction with complex 202, thus releasing the second strand (A-
Such an “mRNA adaptor” does not just sense an mRNA molecule. It can sense any single-stranded nucleic acid, including without limitation viral DNA and mutated tumor DNA.
Accordingly, the present disclosure provides a composition comprising a first nucleic acid complex (e.g., 202 in
Further, the second complex (203) comprises (a) a first strand comprised of, sequentially, a first fragment (P), a second fragment (B) and a third fragment (Ā) that have suitable sequence complementarity to the first (A), second (
Chemical modifications to nucleic acid fragments that can be exposed as single strands are contemplated, as illustrated in
In accordance with one aspect of the disclosure, one embodiment of the disclosure provides a composition comprising: (a) a first nucleic acid complex comprising (i) a first nucleic strand comprising, sequentially, a first, second and third fragments and defined as S-Q-P, wherein each letter denotes a fragment and a string of letters connected by “-” denotes a strand, and (ii) a second strand comprising, sequentially, a first, second, third and fourth fragments and defined as A-
In one aspect, the first strand (S-Q-P-R) of the first complex has suitable sequence complementarity to a fragment on a nucleic acid of a pathogenic nucleic acid to allow binding therebetween under hybridizing conditions.
In one aspect, the pathogenic nucleic acid is a viral DNA, a viral RNA, a bacterial DNA, a bacterial RNA, a mutant tumor DNA, or a tumor RNA.
II. RNAi Adaptor
Further provided is an adaptor that takes a nucleic acid as input and an RNAi (e.g., siRNA) molecule as output, so that the siRNA, upon release, can carry out the intended biological functions. Such an adaptor is illustrated in
In
Complex 303 is similar to translators 102-104 in
Therefore, upon release of the distal (i.e., right hand side) half of complex 302 as a new dual toe-hold, the new toe-hold initiates a strand displacement reaction with complex 303, resulting in the release of a complex includes the distal half of the complex along with the RNAi strands. Such a new complex then can initiate a strand displacement reaction with complex 304, leading to release of all of the RNAi molecules (
Thus, in on embodiment, provided is a composition comprising a first nucleic acid complex comprising (a) a first, a second, a third and a fourth nucleic acid strands each comprising, sequentially, a first, a second and a third fragments and (b) a first and second RNA strands, wherein the first complex comprises: (i) a first duplex region formed between the second fragments of the first and second strands; (ii) a second duplex region formed between the second fragments of the third and fourth strands; (iii) a third duplex region formed between the third fragment of the first strand and the first fragment of the third strand; (iv) a fourth duplex region formed between the first fragment of the second strand and the third fragment of the fourth strand; (v) a fifth duplex region formed between the third fragment of the third strand and the first RNA; and (vi) a sixth duplex region formed between the first fragment of the fourth strand and the second RNA, and the first fragment (
In one aspect, the composition further comprises a second nucleic acid complex comprising a first and a second nucleic strands and a first and a second RNA strands, wherein: the second complex comprises a first duplex region formed between the second fragments of the first and second strands, a second duplex region formed between the third fragment of the first strand and the first RNA strand, and a third duplex region formed between the first fragment of the second strand and the second RNA strand, wherein the first fragment (E) of the first strand and the third fragment (
III. Antisense Adaptor
An adaptor also is provided for receiving signals from upstream translators and releasing an antisense RNA. With reference to
Upon release from the complex 402 by a strand displacement reaction initiated by toe-hold 401, therefore, the distal half of complex (402), which is the new dual toe-hold, targets complex 403 and releases a partial duplex that includes F-Z and
Upon contact with a cellular RNA molecule that includes an E-Z fragment, therefore, the
A unique advantage of such a design is that the
In the present method, therefore, an antisense nucleic acid of a length of greater than, e.g., 30 nt, 35, nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 80 nt, 90 or 100 nt, can be suitably introduced into a biological system.
Thus, one embodiment provides a composition comprising: (a) a first nucleic acid complex comprising (i) a first nucleic acid strand comprising, sequentially, a first, second and third fragments and defined as B-X-D, (ii) a second nucleic acid strand comprising, sequentially, a first, second and third fragments and defined as
In some aspects, the first fragment (F) of the fourth strand of the first complex is at least 5 nucleotides (nt), or 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt longer than the third fragment (Ē) of the third strand of the first complex. In some aspects, the first fragment (
IV. Fan-In
Also provided is a “fan-in” translator set. A fan-in translator set can take input from two or more different nucleic acids as input signal and produce the same output. In other words, a fan-in translator set can carry out an OR operation. A fan-in set is illustrated in
In the illustrated fan-in translator set, translators 502-503 and 505-506 are each similar to 102-103 in
Whether the input is toe-hold 501 or 504, therefore, a strand displacement reaction at translator 507 will be triggered.
Accordingly, in one embodiment, provided is a composition comprising a first and a second nucleic acid complexes each comprising a first, a second, a third, and a fourth nucleic acid strands, each of the strands comprising, sequentially, a first, a second and a third fragments, wherein the nucleic strands are defined as: B-X-D,
V. Fan-Out
Opposite to a fan-in translator set, a “fan-out” translator set takes a single input and produce multiple output nucleic acids. In one aspect, the multiple output nucleic acids have difference sequences so that they can carry out different functions or downstream reactions. In another aspect, the multiple output nucleic acids are identical and thus the fan-out translator set serves as an amplifier.
In
The translators in
The translator set in
Similar to the translator set in
One embodiment of the disclosure, therefore, provides a nucleic acid complex comprising a first, a second, a third, a fourth, a fifth and a sixth nucleic acid strands, wherein: the first strand comprises, sequentially, a first, a second, a third and a fourth fragments and is defined as
Likewise, a nucleic acid complex that fans out two or more different toe-holds is also provided.
VI. Compound Conditional
A “compound conditional” translator set carries out an AND operation. A compound conditional set requires two separate input signals to activate and release an output nucleic acid.
Translator 705 contains at least six strands comprised, from left to right, of three duplex pairs. Only upon contact with the first new toe-hold released from translator 703, can translator 705 undergo a strand displacement reaction, resulting in exposure of two single-stranded regions (
Subsequently, upon contact with the second new toe-hold produced by translator 704, another strand displacement reaction occurs and releases the third duplex pair of 705 as a third new toe-hold. This third new toe-hold then can optionally trigger further reactions through translator 706, for instance.
Thus, in one embodiment, provided is a composition comprising a first, a second and a third nucleic acid complexes, wherein: the first complex comprises a first, a second, a third and a fourth nucleic acid strands each comprising, sequentially, a first, a second and a third fragments and defined as B-W-D,
VII. Inverter
An “inverter” constitutes one or more translators which, upon contact with an input signal, produce a nucleic acid (“stopper”) that blocks a strand displacement reaction that otherwise would take place upon initiation by a different input signal in the system.
As illustrated in
Nucleic acid complex 803 is a translator that takes toe-hold 801 as input and produces the distal half of the complex, a new toe-hold, as output. This new toe-hold, if in contact with translator 805, can carry out a subsequent strand displacement reaction, with potential subsequence reaction with translator 806.
The signal processing route of 801-803-805-806 cannot happen, however, if toe-hold 802 comes into contact with inverter 804 and releases the stopper strand C-
Provided in embodiment, therefore, is a composition comprising a first and a second nucleic complexes, wherein: the first complex comprises a first, a second, a third and a fourth nucleic acid strands each comprising, sequentially, a first, a second and a third fragments and defined as B-X-D,
For any of the above translators and translator sets, it is contemplated that chemical modifications can be used to reduce or eliminate toe-hold clashing, that is, unintended binding between toe-holds and single-stranded regions of any translators in a system, such as a cell. Such modifications can easily be designed with information provided in the disclosure. Further, for each translator set, the corresponding illustrative figure provides exemplary modifications, as annotated by subscripts N, R, and L. The desired properties of such modifications are provided in Table 2.
In some aspects, it is noted that nucleic acid fragments, if not specifically designated as a part of a duplex region, are meant to be single-stranded. Indication of single-strandedness is also apparent in the accompanying figures.
The length of each nucleic acid strand or fragment can be determined computationally or experimentally. Table 1 illustrates the influence of such length, in particular for toe-holds. In one aspect, each of the fragments is from about 3 bases to about 50 bases long. Alternatively, the fragments, are at least about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 bases long. In another aspect, the fragments are not longer than about 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 28, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 bases. Also, fragments that exists in single-stranded forms, such as the toe-holds, can be relatively shorter, such as between about 3, 4, 5, 6, 7, 8, 9, 10 bases and about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 28, 17, 16, or 15 bases. In one aspect, a toe-hold fragment is at least about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 bases long. In another aspect, a toe-hold fragment is not longer than about 20, 19, 28, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 bases. By contrast, the fragments that form duplex regions within a translator can be relatively longer.
The present disclosure further provides methods of using translators of the present disclosure to conduct computation and, in particular, propagate information in a biological environment. For instance, an mRNA adaptor can be used to detect the presence of a pathogenic or neoplastic nucleic acid and produces an output signal in the form of, e.g., a toe-hold. If necessary, multiple mRNA adaptors can be used having specificity to multiple nucleic acids to ensure reliable detection.
Other translators in the system, such as fan-in, fan-out, and inverter translators, then can be used to process the signals generated by the mRNA adaptors, leading eventually to productions of regulatory nucleic acids, e.g., RNAi and antisense RNA, with the corresponding RNAi and antisense adaptor. By virtue of carefully tailored processing, the exact sequences and amounts of these regulatory RNA can be controlled with precision. Accordingly, biological reactions such as apoptosis and immune response can be triggered by these regulatory RNA, bringing about therapeutic or diagnostic benefits.
It would be appreciated readily that the translators, compositions, and systems described here can include biological materials, but they also can be modeled mathematically with a computer. Accordingly, the present disclosure also provides computer methods, systems, and program code-embedded media for carrying out such modeling.
Corresponding to each translator, composition or system in the present disclosure, therefore, a computer-implemented method is provided in which the computer is configured to represent their sequence, structure, synthesis, and strand displacement reaction.
The methodology described here can be implemented on a computer system or network. A suitable computer system can include at least a processor and memory; optionally, a computer-readable medium that stores computer code for execution by the processor. Once the code is executed, the computer system carries out the described methodology.
In this regard, a “processor” is an electronic circuit that can execute computer programs. Suitable processors are exemplified by but are not limited to central processing units, microprocessors, graphics processing units, physics processing units, digital signal processors, network processors, front end processors, coprocessors, data processors and audio processors. The term “memory” connotes an electrical device that stores data for retrieval. In one aspect, therefore, a suitable memory is a computer unit that preserves data and assists computation. More generally, suitable methods and devices for providing the requisite network data transmission are known.
Also contemplated is a non-transitory computer readable medium that includes executable code for carrying out the described methodology. In certain embodiments the medium further contains data or databases needed for such methodology.
The present disclosure is further illustrated by reference to this example, which presents the results of kinetic simulation of the time course behavior of Rotationally Sequestered Translator (RST) networks of the present disclosure, as compared side by side to kinetic simulations of the Toe-Hold Sequestered Translator (THST) networks, mentioned above under the subsection heading “Toe-Hold Sequestering,” with reference to
Mechanisms were generated that included series of N translations, referred to as “the number of stages,” which derive their forward rate constants from Frezza, B. M., O
For each simulation, foreground signal and background signal simulations were conducted for both networks of RSTs and THSTs. “Foreground signal” refers to the concentration of final output sequence generated, given initial input sequence at equal concentration to the other translators in the simulation. “Background signal” denotes the concentration of final output sequence generated, given no initial input sequence.
These simulations demonstrate the idealized response each network design should provide under conditions of varying toe-old length, concentration of the translators, and size of the network (number of stages).
In another series of simulations, an 8 nt toe-hold at 100 nm translator concentrations produced similar foreground signals for both RSTs and THSTs. The systems were examined with increasing depth of the network, in terms of number of stages, i.e., the number of translators. As shown in
Yet another series of simulations examined moderate sized networks (5 stages), with both long (13 nt) and short (8 nt) toe-holds, and showed how their behavior changed with variation in concentration of the translators. The results are presented in
In the case of long toe-holds, reaction timescales remained biologically relevant for RST to far lower concentrations, which were still showing modest reactivity down at 100 pM, whereas THST required much higher concentration (10 μM) to obtain any meaningful activity (
Together these data demonstrate that RSTs offer design possibilities whereby background (leak) activity can be reduced to nearly undetectable levels with longer toe-hold, without losing reactivity through extreme inhibition as the THSTs face. Furthermore, they show that RSTs can function with ideal foreground-to-background reactivity in an enormous dynamic range of concentrations, suiting them ideally for use under biological circumstances.
While particular embodiments of the subject invention have been discussed, they are illustrative only and not restrictive of the invention. A review of this specification will make many variations of the invention apparent to those skilled in the field of the invention. The full scope of the invention should be determined by reference both to the claims below, along with their full range of equivalents, and to the specification, with such variations.
This application is a continuation of U.S. application Ser. No. 16/664,713, filed Oct. 25, 2019, now U.S. Pat. No. 10,883,106, which is a continuation of U.S. application Ser. No. 15/470,754, filed Mar. 27, 2017, now U.S. Pat. No. 10,479,994, which is a divisional of U.S. application Ser. No. 14/721,398, filed May 26, 2015, now U.S. Pat. No. 9,624,495, which is a divisional of U.S. application Ser. No. 13/801,762, filed Mar. 13, 2013, now U.S. Pat. No. 9,068,218, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/754,339, filed Jan. 18, 2013, the contents of each of which are incorporated here by reference in their entireties.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6238927 | Abrams et al. | May 2001 | B1 |
| 7501253 | Pourmand et al. | Mar 2009 | B2 |
| 7538202 | Zhang et al. | May 2009 | B2 |
| 9068218 | Kleinbaum et al. | Jun 2015 | B2 |
| 9624495 | Kleinbaum et al. | Apr 2017 | B2 |
| 10479994 | Kleinbaum et al. | Nov 2019 | B2 |
| 20020119458 | Suyama et al. | Aug 2002 | A1 |
| 20030152924 | Ullman et al. | Aug 2003 | A1 |
| 20050075792 | Shapiro et al. | Apr 2005 | A1 |
| 20050112065 | Drummond et al. | May 2005 | A1 |
| 20070072215 | Seelig et al. | Mar 2007 | A1 |
| 20090170719 | Kazakov et al. | Jul 2009 | A1 |
| 20090191546 | Zhang et al. | Jul 2009 | A1 |
| 20110294687 | Kleinbaum | Dec 2011 | A1 |
| 20110306758 | Zhang | Dec 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-2008097929 | Aug 2008 | WO |
| WO-2008141279 | Nov 2008 | WO |
| WO-2012151328 | Nov 2012 | WO |
| Entry |
|---|
| Allawi et al., “Thermodynamics and NMR of Internal G-T Mismatches in DNA.” Biochemistry vol. 36, (1997):10581-10594. |
| Anonymous, “DNA nanotechnology”, Wikipeida, the free encyclopedia, 2012, https://en.wikipedia.org/wiki/DNA_nanotechnology?oldid=527955533, pp. 1-14. |
| Beaucage, S. L.; Uyer, R. P., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Tetrahedron, 1992, 48, 2223-2311. |
| Beck et al., “Solid Phase Synthesis of PNA Oligomers,” Methods In Molecular Biology, vol. 208, pp. 29-41 (2009) Abstract. |
| Biswas et al., “Branch Migration Through DNA Sequence Heterology” J. Mol. Biol., (1998) 279, 795-806. |
| Braasch et al., “Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA,” Chemistry & Biology, 8 (2001) 1-7. |
| Brown et al., “A Brief History of Oligonucleotide Synthesis”, Methods in Molecular Biology, pp. 1-17 (1993). |
| Corradini et al., “Control of helical handedness in DNA and PNA nanostructures,” Methods in Molecular Biology, 749:79-92 (2011). |
| Doktycz et al., “Nucleic Acids: Thermal Stability and Denaturation,” Encyclopedia of Life Sciences, pp. 1-18, John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003123] (Oct. 2002). |
| Dragulescu-Andrasi et al., “A simple gamma-backbone modification preorganizes peptide nucleic acid into a helical structure”, Journal of the American Chemical Society, 2006, vol. 128, pp. 10258-10267. |
| Dragulescu-Andrasi et al., “Cell-permeable GPNA with appropriate backbone stereochemistry and spacing binds sequence-specifically to RNA,” Chem. Commun., 2005, pp. 244-246. |
| Duose et al., “Configuring robust DNA strand displacement reactions for in situ molecular analyses”, Nucleic Acids Research, vol. 40, No. 7, pp. 3289-3298 (2012). |
| Extended European search report for European Application No. 14740468.5, dated Nov. 15, 2016. (15 pages). |
| Freier et al., “The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes”, Nucleic Acids Research, 1997, vol. 25, No. 22,, pp. 4429-4443. |
| Frezza et al., “Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates”, J. Am. Chem. Soc., 2007, 129, pp. 14875-14879. |
| International Search Report and Written Opinion for International Application No. PCT/US2014/011782, dated May 22, 2014. |
| International Preliminary Report on Patentability for International Application No. PCT/US2014/011782, dated Jul. 21, 2015. (5 pages). |
| Iyer et al., “Oligonucleotide Synthesis”, Comprehensive Natural Products Chemistry vol. 7 (DNA and Aspects of Molecular Biology), 1999, pp. 105-152. |
| Kahan et al., “Towards molecular computers that operate in a biological environment”, Science Direct Physica D 237, (2008) 1165-1172. |
| Krane et al., “Time for DNA Disclosure”, Science, vol. 326, Dec. 18, 2009, 1631-1633. |
| Kutyavin et al., “Oligonucleotides Containing 2-Aminoadenine and 2-Thiothymine Act as Selectively Binding Complementary Agents,” Biochemistry (1996) 35:11170-11176. |
| Lakin et al., “Design and Analysis of DNA Strand Displacement Devices Using Probabilistic Model Checking”, Journal of The Royal Society Interface, 9(72): 1470-1485 (especially pp. 1481-1484) (2012). |
| Laplanche et al., “Phosphorothioate-Modified Oligodeoxyribonucleotides. III. NMR and UV Spectroscopic Studies of the Rp-Rp, Sp-Sp and Rp-Sp, Duplexes (d(GGSAATTCC))2, Derive from Diastereomeric O-Ethyl Phosphorothioates”, Nucleic Acids Research, 14(22):9081-9093 (1986). |
| Latimer et al., “Synthetic repeating sequence DNAs containing phosphorothioates: nuclease sensitivity and triplex formation,” Nucleic Acids Res. 17(4):1549-1561 (1989). |
| Lee et al., “Chitosan: a novel platform in proton-driven DNA strand rearrangement actuation,” Mol. BioSyst. (2009) 5:391-396. |
| Li et al., “A new class of homogeneous nucleic acid probes based on specific displacement hybridization.” Nucleic Acids Research, vol. 30, No. 2, pp. E5.1-E5.9 (2002). |
| Lusvarghi et al., “Loop and backbone modifications of peptide nucleic acid improve G-Quadruplex binding selectivity”, JACS, 131, 18415-18424, (2009). |
| Ma et al., “3-Arm Nucleic Acid Junctions are Flexible”, Nucleic Acids Research, 14:9745-9753 (1986). |
| Maugh II, “Wolf & Lamb Chemistry, Many useful reactions difficult to perform by conventional means can be carried out easily with polymeric reagents”, Science, vol. 217, Aug. 20, 1982, pp. 719-720. |
| Nielsen et al., “An Introduction to Peptide Nucleic Acid”, Current Issues Molec. Biol. (1999), 1(2):89-104. |
| Nishikawa et al., “DNA computation simulator based on abstract bases”, Soft Computing 5:25-38 (2001). |
| Ortega et al., “Binding Affinities of Oligonucleotides and PNAs Containing Phenoxazine and G-Clamp Cytosine Analogues are Unusually Sequence Dependent”, Organic Letters, 2007, vol. 9, No. 22, 4503-4506. |
| Panyutin et al., “Formation of a Single Base Mismatch Impedes Spontaneous DNA Branch Migration”, J. Mol. Biol., 230:413-424 (1993). |
| Panyutin et al., “The kinetics of spontaneous DNA branch migration”, Proceedings of the National Academy of Sciences of USA, vol. 91, Mar. 1994, pp. 2021-2025. |
| Partial supplementary European search report for European Application No. 14740468.5, dated Aug. 12, 2016. (10 pages). |
| Picuri et al., “Universal Translators for Nucleic Acid Diagnosis”, J. Am. Chem. Soc. (2009) 131:9368-9377. |
| Sager et al., “Designing nucleotide sequences for computation: A survey of constraints”, DNA11, LNCS 3892: 275-289, (2006). |
| Sahu et al., “Synthesis of Conformationally Preorganized and Cell Permeable Guanidine-Based Peptide Nucleic Acids (GPNAs)”, J. Org. Chem. 2009, 74, 1509-1516. |
| Seelig et al., “Enzyme-Free Nucleic Acid Logic Circuits”, Science, vol. 314, Dec. 8, 2006, pp. 1585-1588. |
| Seeman et al., “DNA Branched Junctions”, Annu. Rev. Biophys. Biomol. Struct. 1994. 23:53-86. |
| Seeman et al., “Nanotechnology and the double helix”, Scientific American, 2004, vol. 290, No. 6, pp. 64-75. |
| Summerton et al., “Morpholino Antisense Oligomers: Design, Preparation, and Properties”, Antisense & Nucleic Acid Drug Development 7:187-195 (1997). |
| Tajima et al., “Direct Oxidative Cyanation Based on the Concept of Site Isolation”, J. Am. Chem. Soc. 2008, 130, 10496-10497. |
| Uhlmann et al., “Synthesis and Properties of PNA/DNA Chimeras”, Angew. Chem. Ed. Engl. 35(22):2632-2635 (1996). |
| Voelcker et al., “Sequence-Addressable DNA Logic”, Small 2008, 4, No. 4, pp. 427-431. |
| Voit et al., “Sequential One-Pot Reactions Using the Concept of Site Isolation”, Angewandte Chemical International Edition (2006) 45:4238-4240. |
| Yashin et al., “Networking Particles over Distance Using Oligonucleotide-Based Devices”, J. Am. Chem. Soc. (2007) 129:15581-15584. |
| Zhang et al., “Control of DNA Strand Displacement Kinetics Using Toehold Exchange”, J. Am. Chem. Soc. (2009) 131:17303-17314. |
| Zhang et al., “Dynamic DNA nanotechnology using strand-displacement reactions”, Nature Chemistry, vol. 3, No. 2, pp. 103-113 (2011). |
| Zhang et al., “Engineering entropy-driven reactions and networks catalyzed by DNA”, Science, vol. 318, Nov. 2007, pp. 1121-1125. |
| Zhou et al., “Novel Binding and Efficient Cellular Uptake of Guanidine-Based Peptide Nucleic Acids (Gpna)”, J. Am. Chem. Soc., 2003, 125:23, 6878-6879. |
| Number | Date | Country | |
|---|---|---|---|
| 20210102207 A1 | Apr 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61754339 | Jan 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14721398 | May 2015 | US |
| Child | 15470754 | US | |
| Parent | 13801762 | Mar 2013 | US |
| Child | 14721398 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16664713 | Oct 2019 | US |
| Child | 17107699 | US | |
| Parent | 15470754 | Mar 2017 | US |
| Child | 16664713 | US |
