U.S. patent application number 16/875641 was filed with the patent office on 2021-02-04 for system and method for propagating information using modified nucleic acids.
The applicant listed for this patent is Emerald Therapeutics, Inc.. Invention is credited to Daniel J. Kleinbaum.
Application Number | 20210034944 16/875641 |
Document ID | / |
Family ID | 1000005152333 |
Filed Date | 2021-02-04 |
United States Patent
Application |
20210034944 |
Kind Code |
A1 |
Kleinbaum; Daniel J. |
February 4, 2021 |
SYSTEM AND METHOD FOR PROPAGATING INFORMATION USING MODIFIED
NUCLEIC ACIDS
Abstract
A method is provided for improving a nucleic acid-based
molecular computing system comprised of (i) a nucleic acid
structure, (ii) at least one polynucleotide displacement molecule
that can bind with the nucleic acid structure under hybridizing
conditions, and (iii) a clashing polynucleotide molecule that
competes with the polynucleotide displacement molecule for binding
the nucleic acid structure under the hybridizing conditions The
method entails incorporation of chemical modification that inhibits
the binding of the clashing molecule and the nucleic acid structure
or facilitates the binding of the displacement molecule and the
nucleic structure.
Inventors: |
Kleinbaum; Daniel J.;
(Redwood City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emerald Therapeutics, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000005152333 |
Appl. No.: |
16/875641 |
Filed: |
May 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15375629 |
Dec 12, 2016 |
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16875641 |
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14142531 |
Dec 27, 2013 |
9547750 |
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15375629 |
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13072438 |
Mar 25, 2011 |
8630809 |
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14142531 |
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61349012 |
May 27, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6837 20130101;
G06N 3/002 20130101; B82Y 10/00 20130101; G06N 3/123 20130101; G16B
30/00 20190201 |
International
Class: |
G06N 3/00 20060101
G06N003/00; G16B 30/00 20060101 G16B030/00; B82Y 10/00 20060101
B82Y010/00; G06N 3/12 20060101 G06N003/12; C12Q 1/6837 20060101
C12Q001/6837 |
Claims
1. A method for improving a nucleic acid-based molecular computing
system, comprising (A) identifying a computing system comprised of
(i) a nucleic acid structure that comprises an incompletely
base-paired duplex domain, (ii) at least one polynucleotide
displacement molecule that can bind with said nucleic acid
structure under hybridizing conditions, such that said nucleic acid
structure undergoes a transition in energy state due to a branch
migration reaction involving said duplex domain, and (iii) a
clashing polynucleotide molecule that competes with said
polynucleotide displacement molecule for binding said nucleic acid
structure under said conditions but that cannot produce a branch
migration reaction involving said duplex domain; then (B)
reconfiguring at least one of said displacement molecule and said
nucleic acid structure, respectively, to incorporate a chemical
modification relative to a first reference molecule that comprises
natural nucleotides and has the same sequence content as said
displacement molecule or said nucleic acid structure, as the case
may be, wherein said modification causes binding of said
displacement molecule and said nucleic acid structure to have a
hybridization free energy, differing from that of a first reference
binding between said displacement molecule or said nucleic acid
structure and said first reference molecule, such that said branch
migration reaction is facilitated relative to said first reference
binding; and/or (C) reconfiguring at least one of said clashing
molecule and said nucleic acid structure, respectively, to
incorporate a chemical modification relative to a second reference
molecule that comprises natural nucleotides and has the same
sequence content as said clashing molecule or said nucleic acid
structure, as the case may be, wherein said modification causes
binding of said clashing molecule and said nucleic acid structure
to have a hybridization free energy, differing from that of a
second reference binding between said clashing molecule or said
nucleic acid structure and said second reference molecule, such
that binding of said clashing molecule is impeded relative to said
second reference binding.
2. The method of claim 1, wherein said branch migration reaction is
facilitated by: increasing a probability of productive reactions in
which binding of said nucleic acid structure by said displacement
molecule has, by virtue of said chemical modification, a lower
hybridization free energy than does a first reference binding
reaction; or
3. The method of claim 1, wherein said branch migration reaction is
facilitated by: reducing a probability of unproductive reactions in
which binding of said nucleic acid structure by said clashing
molecule has, by virtue of said chemical modification, a higher
hybridization free energy than does a second reference binding
reaction.
4. The method of claim 1, wherein said nucleic acid structure is
part of an enzyme-free, nucleic acid logic gate selected from the
group consisting of an AND gate, a NOT gate, an OR gate, a NAND
gate, a NOR gate, an XOR gate, and an XNOR gate.
5. The method of claim 1, wherein said chemical modification is
selected from the group consisting of (i) replacing the
sugar-phosphodiester backbone of said nucleotides with a
pseudo-peptide backbone, (ii) modifying the sugar moiety of said
nucleotides, and (iii) substituting an analogue for the nitrogenous
base of at least one of said nucleotides.
6. The method of claim 5, wherein said chemical modification
comprises replacing the sugar-phosphodiester backbone of said
nucleotides with a pseudo-peptide backbone into which a guanidinium
functional group is incorporated.
7. The method of claim 5, wherein said chemical modification
comprises substituting a tricyclic cytosine analogue for the
nitrogenous base of at least one of said nucleotides.
8. The method of claim 5, wherein said chemical modification
comprises introducing a heteroatom at the 2'-position of said sugar
moiety.
9. The method of claim 1, wherein said branch migration reaction
comprises a toe-hold-mediated strand displacement.
10. A nucleic acid-based system for propagating information, the
system comprising: (A) a nucleic acid structure that comprises an
incompletely base-paired duplex domain; (B) at least one
polynucleotide displacement molecule to effect binding with said
nucleic acid structure under hybridizing conditions, such that said
nucleic acid structure undergoes a transition in energy state due
to a branch migration reaction involving said duplex domain; and
(C) at least one polynucleotide clashing molecule capable of
binding with said nucleic acid structure under hybridizing
conditions, such that said nucleic acid structure and said clashing
molecule are bound and prevent the binding of the polynucleotide
displacement molecule, wherein at least one of said displacement
molecule and said nucleic acid structure, respectively, has a
chemical modification relative to a reference molecule that
comprises natural nucleotides and has the same sequence content as
said displacement molecule or said nucleic acid structure, as the
case may be, wherein said modification causes said binding to have
a hybridization free energy, differing from that of a reference
binding between said displacement molecule or said nucleic acid
structure and a reference molecule, such that said branch migration
reaction is facilitated relative to said reference binding, and/or
wherein at least one of said clashing molecule and said nucleic
acid structure, respectively, has a chemical modification relative
to a reference molecule that comprises natural nucleotides and has
the same sequence content as said displacement molecule or said
nucleic acid structure, as the case may be, wherein said
modification causes said binding to have a hybridization free
energy, differing from that of a reference binding between said
displacement molecule or said nucleic acid structure and a
reference molecule, such that binding of said clashing molecule is
impeded relative to said reference binding.
11. The system of claim 10, wherein said nucleic acid structure is
part of an enzyme-free, nucleic acid logic gate selected from the
group consisting of an AND gate, a NOT gate, an OR gate, a NAND
gate, a NOR gate, an XOR gate, and an XNOR gate.
12. The system of claim 11, wherein said system comprises one or
more circuits comprised of a plurality of enzyme-free, nucleic acid
logic gates.
13. The system of claim 10, wherein said chemical modification is
selected from the group consisting of (i) replacing the
sugar-phosphodiester backbone of said nucleotides with a
pseudo-peptide backbone, (ii) modifying the sugar moiety of said
nucleotides, and (iii) substituting an analogue for the nitrogenous
base of at least one of said nucleotides.
14. The system of claim 13, wherein said chemical modification
comprises replacing the sugar-phosphodiester backbone of said
nucleotides with a pseudo-peptide backbone into which a guanidinium
functional group is incorporated.
15. The system of claim 14, wherein said chemical modification
comprises substituting a tricyclic cytosine analogue for the
nitrogenous base of at least one of said nucleotides.
16. The system of claim 14, wherein said chemical modification
comprises introducing a heteroatom at the 2'-position of said sugar
moiety.
17. The system of claim 10, wherein said branch migration reaction
comprises a toe-hold-mediated strand displacement.
18. A system for propagating information, the system comprising a
first molecule, anda second molecule configured to bind with the
first molecule, wherein (A) at least one of the first molecule or
the second molecule, respectively, has a chemical modification
relative to a reference molecule that has the same sequence content
as the first molecule or the second molecule, as the case may be,
and (B) the modification causes said binding to have a free energy
different from that of a reference binding between the reference
molecule and the first or second molecule, such that at least one
of the following is realized in the system: (i) the probability of
a productive binding is raised; and (ii) the probability of an
unproductive binding is reduced.
19. The system of claim 18, wherein the productive binding
comprises a binding that propagates information, and wherein the
unproductive binding comprises a binding that does not propagate
information.
20. The system of claim 18, wherein the free energy difference as a
result of the modification decreases for the productive binding,
and increases for the unproductive binding.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/375,629, filed Dec. 12, 2016, which is a
continuation of U.S. patent application Ser. No. 14/142,531, filed
Dec. 27, 2013, now Pat. No. 9,547,750, issued Jan. 17, 2017, which
is a divisional of U.S. patent application Ser. No. 13/072,438,
filed Mar. 25, 2011, now Pat. No. 8,630,809, issued Jan. 14, 2014,
which claims priority to U.S. Provisional Application No.
61/349,012, filed May 27, 2010, the entire contents of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Nano-scale computers can potentially be realized using
molecules. Such computers may be suited for solving certain
computation problems. In particular, computers employing
biomolecules can be compatible with biological environments, and
can potentially be used for complex disease diagnostics or even
treatments.
[0003] The ability to translate one nucleic acid sequence into
another can be employed to build logic gates and networks with
nucleic acids. These gates and networks are driven by two events:
hybridization and strand displacement. Both events are generally
thermodynamically favorable; that is, they involve a transition
from a higher to a lower-energy state. Thus, both events can occur
spontaneously in a system.
[0004] Hybridization involves free, single-stranded stretches of
nucleic acids. Accordingly, a nucleic-acid network may be regulated
by the availability of these free strands.
[0005] 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.
[0006] These translators are the foundation on which basic logic
operators, such as AND, NOT, OR, NAND, NOR, XOR and XNOR, can be
built with nucleic acids. From these and other logic components,
larger networks can be constructed that include components such as
amplifiers. As a result, these translation events are important for
information processing with nucleic acids and molecular
computing.
SUMMARY OF THE INVENTION
[0007] In accordance with one aspect of the present invention, a
method is provided for improving a nucleic acid-based molecular
computing system. The method includes: (A) identifying a computing
system comprised of (i) a nucleic acid structure that includes an
incompletely base-paired duplex domain, (ii) at least one
polynucleotide displacement molecule that can bind with the nucleic
acid structure under hybridizing conditions, such that the nucleic
acid structure undergoes a transition in energy state due to a
branch migration reaction involving the duplex domain, and (iii) a
clashing polynucleotide molecule that competes with the
polynucleotide displacement molecule for binding the nucleic acid
structure under the hybridizing conditions but that cannot produce
a branch migration reaction involving the duplex domain; then (B)
reconfiguring at least one of the displacement molecule and the
nucleic acid structure, respectively, to incorporate a chemical
modification relative to a first reference molecule that comprises
natural nucleosides and has the same sequence content as the
displacement molecule or the nucleic acid structure, as the case
may be. The aforementioned the modification causes binding of the
displacement molecule and the nucleic acid structure to have a
hybridization free energy, differing from that of a first reference
binding between the displacement molecule or the nucleic acid
structure and the first reference molecule, such that the branch
migration reaction is facilitated relative to the first reference
binding. After or in place of step (B) is a step (C) of
reconfiguring at least one of the clashing molecule and the nucleic
acid structure, respectively, to incorporate a chemical
modification relative to a second reference molecule that comprises
natural nucleosides and has the same sequence content as the
clashing molecule or the nucleic acid structure, as the case may
be. The modification causes binding of the clashing molecule and
the nucleic acid structure to have a hybridization free energy,
differing from that of a second reference binding between the
clashing molecule or the nucleic acid structure and the second
reference molecule, such that binding of the clashing molecule is
impeded relative to the second reference binding.
[0008] The invention also provides, in another of its aspects, a
system that includes (A) a nucleic acid structure that comprises an
incompletely base-paired duplex domain; (B) at least one
polynucleotide displacement molecule to effect binding with the
nucleic acid structure under hybridizing conditions, such that the
nucleic acid structure undergoes a transition in energy state due
to a branch migration reaction involving the duplex domain; and (C)
at least one polynucleotide clashing molecule capable of binding
with the nucleic acid structure under hybridizing conditions, such
that the nucleic acid structure and the clashing molecule are bound
and prevent the binding of the polynucleotide displacement
molecule. At least one of the displacement molecule and the nucleic
acid structure, respectively, has a chemical modification relative
to a reference molecule that comprises natural nucleosides and has
the same sequence content as the displacement molecule or the
nucleic acid structure, as the case may be. The modification causes
the binding to have a hybridization free energy, differing from
that of a reference binding between the displacement molecule or
the nucleic acid structure and a reference molecule, such that the
branch migration reaction is facilitated relative to the reference
binding. In addition or in the alternative, at least one of the
clashing molecule and the nucleic acid structure, respectively, has
a chemical modification relative to a reference molecule that
comprises natural nucleosides and has the same sequence content as
the displacement molecule or the nucleic acid structure, as the
case may be. The modification causes the binding to have a
hybridization free energy, differing from that of a reference
binding between the displacement molecule or the nucleic acid
structure and a reference molecule, such that binding of the
clashing molecule is impeded relative to the reference binding.
[0009] In yet another aspect, a system is provided for propagating
information. The system includes a first molecule and a second
molecule configured to bind with the first molecule. At least one
of the first molecule and the second molecule, respectively, has a
chemical modification relative to a reference molecule that has the
same sequence content as the first molecule or the second molecule,
as the case may be. The modification causes the binding to have a
free energy different from that of a reference binding between the
reference molecule and the first or second molecule, such that at
least one of the following is realized in the system: a probability
of a productive binding is raised, or a probability of an
unproductive binding is reduced.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A illustrates a solid-phase sequestering
implementation of a nucleic acid translator, where individual
section (A, B, etc) represent stretches of oligonucleotides of
arbitrary length and sequence.
[0011] FIG. 1B illustrates how a solid-phase sequestering
implementation of a nucleic acid translator sequesters stretches of
oligonucleotides.
[0012] FIG. 2A illustrates a "toe-hold" sequestering implementation
of a nucleic acid translator. Again, individual sections represent
stretches of oligonucleotides of arbitrary length and sequence.
[0013] FIG. 2B illustrates how a "toe-hold"-sequestered
implementation of a nucleic acid translator sequesters stretches of
oligonucleotides.
[0014] FIG. 3A shows a system of three toe-hold sequestered nucleic
acid translators. The reactions shown are all strand-displacement
reactions that proceed by the same branch-migration mechanism as in
FIG. 2A.
[0015] FIG. 3B shows a system with the same oligonucleotides as are
shown in FIG. 3A but, instead of strand displacement reactions,
toe-hold "clashes" are shown, where the toe-hold is bound by a
sequence that cannot produce a strand displacement reaction. This
binding event occupies the toe-hold such that the desired strand
cannot bind.
[0016] FIG. 3C illustrates different architectures for incompletely
base-paired nucleic acid structures including: (a) terminal; (b)
internal loop; and (c) and (d) multi-part complex.
[0017] FIG. 4 presents a schematic depiction of how, in the system
of nucleic acid translators shown in FIG. 3A, the equilibrium can
be shifted to favor strand displacement reactions over toe-hold
clashes by replacing the stretches of oligonucleotides with
chemical modifications that increase the binding affinity and/or
the stretches of oligonucleotides with modifications that decrease
the binding affinity.
[0018] FIG. 5 shows modified backbone structures for nucleic acid
analogs, where "B" represents an arbitrary nucelobase, and (a)
shows a natural phosphodiester backbone found in DNA, (b) shows
peptide nucleic acids, (c) shows guanidinium peptide nucleic acids,
(d) shows L-serine derived gamma-PNAs, and (e) shows
phosphorodiamidates (here with a morpholino sugar).
[0019] FIG. 6 illustrates modified sugar structures for nucleic
acid analogs, where "B" represents an arbitrary nucleobase, and (a)
shows a natural deoxyribose sugar found in DNA, (b) shows
morpholinos, (c) shows locked nucleic acids, and (d) shows
fluorine-modified RNA derivative.
[0020] FIG. 7 illustrates modified nucelobase structures for the
nucleic acid analogs methylcytosine (a), diaminopurine (b),
phenoxazine (c), and G-clamp (d).
[0021] FIG. 8 illustrates a fluorescence kinetics assay for
indicating the reaction rate change due to a chemical modification
according to the invention.
DETAILED DESCRIPTION
[0022] Various approaches, such as solid-phase sequestering,
toe-hold sequestering, and toe-hold exchange, can be used in
translating nucleic acid sequences to build logic operators and
networks. These three particular approaches, described in greater
detail below, are exemplified via geometries that utilize
three-way, toe-hold-mediated branch migration reactions. Additional
mechanisms are possible for branch migration reactions, however,
including but not limited to four-way branch migration, four-way
accelerated migration, and multi-strand complex migration.
[0023] Thus, while the following embodiments are described using
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.
[0024] Solid-phase sequestering uses physically separating the
relevant sequences/strands in space, using beads, nanoparticles, or
surfaces to do so. This approach employs principles of site
isolation, which has been used extensively in the organic chemistry
context. In solid-phase sequestered geometries, when these
displacement events occur can be controlled by regulating whether
the necessary strands are in the solution or in the solid phase of
the system.
[0025] FIG. 1A shows a basic solid-phase sequestering setup for a
translator--a component that allows a system to substitute one
nucleic acid sequence for another. Here, the strand A'-X' (where A
and X each represent a stretch of oligonucleotides of arbitrary
length and sequence and X' and A' represent their respective
reverse complements) is bound to a solid support and is initially
hybridized to Y-B-X, forming a nucleic acid structure in the form
of an incompletely base-paired duplex, which can function as a
translator. In this configuration, the strand Y-B-X is solid-phase
sequestered and cannot interact with the rest of the system.
However, in the presence of the strand X-A, referred to as a
"polynucleotide displacement molecule," strand Y-B-X can be
displaced from the solid support and exposed to the solution phase
of the system, while strand X-A is bound to the support. This
operation involves two steps, the first of which is the
hybridization of complementary sequences A and A' (often referred
to as toe-hold binding). In the second step, the X region of strand
X-A binds to the X' region of A'-X', displacing the X region of
Y-B-X and releasing this strand into solution while leaving X-A
bound to the solid support (this step is often referred to as a
branch migration reaction). This two-step process effectively
allows for the translation of a free X-A strand into a free Y-B-X
strand.
[0026] FIG. 1B shows a system having an input strand X-A
interacting with an incompletely base-paired duplex, "Translator
1." The output includes a completely base-paired duplex, A'-X'/X-A,
which is considered a "waste" product, and an output strand Y-B-X,
which is referred to as "Output 1" and can be used as "Input 2" in
a further reaction. "Input 2" interacts with "Translator 2" and
produces the "Output 2" and another waste product. In this figure,
the B region of strand Y-B-X illustrates the sequestering of
sequences in this network. At the start, Y-B-X cannot hybridize
with the B' region of Translator 2 because both are isolated on
separate solid-supports. When Input 1 binds to Translator 1 and
releases Y-B-X into solution, Y-B-X can then interact with
Translator 2. Therefore, the ability of Y-B-X and Translator 2 to
interact is conditional on the presence of Input 1.
[0027] 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.
[0028] Toe-hold sequestering and toe-hold exchange are separate
approaches that use similar pairing interactions, but with
different geometries. Both can accomplish the same operations as
solid-phase translators, but function 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.
For both toe-hold geometries, all of the strands can be in solution
together, by the consequence of which displacement events are
regulated by the availability of toe-holds, i.e., short stretches
of single-strand nucleic acid sequences that provide a starting
point for a displacement event.
[0029] FIG. 2A shows a toe-hold sequestered translator similar to
the one in FIG. 1A, but based on toe-hold rather than solid-phase
sequestering. In this example, the A' region of the translator is
the toe-hold that binds the input strand and allows the strand
displacement reaction to proceed.
[0030] FIG. 2B shows a toe-hold-based system having an input strand
A-X-B interacting with an incompletely base-paired duplex,
"Translator 1." The output includes a waste product, i.e., a
completely base-paired duplex, B'-X'-A'/A-X-B, and "Output 1"
strand X-B-Y-C, which can be used as "Input 2" in a further
reaction. "Input 2" interacts with "Translator 2" and produces the
"Output 2" and another waste product. In this figure, the B region
of X-B-Y-C is sequestered in Translator 1 by being hybridized to a
complementary B' region and therefore unable to interact with the
B' region of Translator 2. The ability of X-B-Y-C to interact with
Translator 2 is conditional on the presence of Input 1 (A-X-B) in
the system.
[0031] The toe-hold geometries have the potential to be very
useful, but their use to date has been limited by the rate at which
a system containing such toe-hold geometries can propagate
information. This is due to inherent limitations, present in the
current toe-hold sequestered approaches, which slow them down below
a biologically useful timescale. These 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.
[0032] FIG. 3A shows a system of three toe-hold sequestered nucleic
acid translators, much like the one in FIG. 2A. If all three
strands are in solution together, however, there are other binding
events that can take place. FIG. 3B illustrates some of the
non-productive binding events or clashes that can take place. By
involving a "polynucleotide clashing molecule," these events do not
lead to a displacement reaction but can slow the system down,
because the incidence of a clashing strand blocks strands from
binding that can produce a displacement reaction.
[0033] Toe-holds can be kept short to mitigate the effect of these
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.
[0034] Pursuant to one aspect of the invention, by virtue of
chemical modification of the nucleic acid structure and/or the
polynucleotide displacement molecule, the productive interactions
are facilitated. This makes it feasible for chaining individual
logic gates together into networks of arbitrary size for biological
applications. More specifically, an approach is provided to
determine how to disfavor clashing interactions and favor
productive interactions without altering the information content of
the sequences.
[0035] In accordance with the invention, these approaches improve
the thermodynamics of binding for the desired strand and/or
disfavor the thermodynamics for the clashing strands, all without
altering the sequence content. In particular, using
chemically-modified structures, the Gibbs free energy (AG) for the
desired hybridization reaction between a given oligonucleotide and
its DNA or RNA complement is reduced and/or the AG of the clashing
interaction is increased. On a molecular level, these respectively
correspond to a tighter binding (higher binding affinity) of
desired hybridization reaction and to a less tight binding (lower
binding affinity) for clashing strands, resulting from the
modification of the structures.
[0036] The former shifts the equilibrium for desired binding events
with the toe-hold toward the duplex (bound) state, creating a
better chance for the displacement reaction to occur without
affecting the equilibrium of binding between the toe-hold and
clashing strands. The latter disfavors the binding of the clashing
strand(s), making these interactions favor the unbound state. These
two changes can be used separately or together, in order to favor
the binding of the desired strand and disfavor the binding of the
clashing strand(s).
[0037] Embodiments in accordance with the invention take advantage
of the fact that the reaction rate of any toe-hold mediated strand
displacement is related to the thermodynamic favorability of the
two nucleic acid strands or complexes being bound. By making these
thermodynamics more favorable for the non-clashing binding events
and/or less favorable for clashing binding events, one can drive
the system toward productive displacement reactions. This will
speed up the rate at which information is propagated to the point
that it can be used on biologically relevant timescales. The
approach of the invention, described here, accomplishes this
through chemical modifications to stretches of oligonucleotides in
the system.
[0038] By virtue of such chemical modification of the nucleic acid
structure and/or of the polynucleotide molecule, pursuant to
invention, the effective concentration range is widened over which
the reaction is optimal. This happens because the percentage of
nucleic acid in a duplex at a given temperature is a function of
concentration and of the AG of the hybridization reaction. Thus,
the more concentrated a set of complementary oligonucleotides is,
the higher the percentage of duplex formation and the lower (more
favorable) the AG is, the higher the percentage of duplex
formation. Changing the AG for a given interaction therefore
changes the percent duplex at a given concentration. Lowering the
AG (i.e., making the interaction more favorable) means that there
will be a higher percentage of duplex at a given concentration,
while raising the AG (making the interaction less favorable) means
there will be a lower percentage of duplex at the same
concentration. This effect widens the optimal concentration range
at which these reactions can be practically used, as desired
duplexes can be formed at lower concentrations and undesired
duplexes will not be formed at higher concentrations, relative to a
reference set of unmodified oligonucleotides.
[0039] These approaches apply to any toe-hold-mediated reaction
geometry, including but not limited to the 3-way branch migration
reactions discussed above. Different architectures can be used for
the incompletely base-paired duplex where branching or loops are
located at different points in the structure (see FIG. 3C, for
instance). The orientation of the branches or loops is not
predicated on the directionality of the strand. Thus, these
structures can include (a) a terminal structure, (b) an internal
loop, or (c) and (d) a multi-part complex, or any other possible
architectures for an incompletely base-paired duplex.
[0040] These nucleic acid structures can translate an active
"input" sequence (the displacement polynucleotide) into an active
"output" sequence (the polynucleotide which is released).
[0041] The nucleic acid structures called "translators" above can
be at their respective thermodynamic minimum; i.e., they are the
most stable structures that the particular set of nucleic acid
sequences can form. These structures can be formed by annealing the
two or more individual nucleic acid strands. For instance, all of
the strands can be mixed together, heated to well above the melting
point for any structure to form, and then slowly cooled down. This
allows the strands to hybridize in the lowest possible energy state
(thermodynamic minimum).
[0042] This procedure can be the same for natural nucleic acids
like DNA and RNA, nucleic acids with modified backbones, sugars, or
bases, and for chimeras made up of natural and modified nucleic
acids.
[0043] Selective incorporation of specific chemical modifications
can favor the binding of strands capable of productive displacement
reactions over toe-hold clash binding events. For example, FIG. 4
shows the same strands as FIG. 3A and 3B. However, if certain
stretches of nucleic acids are replaced with chemically modified
analogs that improve the thermodynamics of binding, the kinetics
for clashes shifts in favor of the displacement reactions. For
example, if the "B" stretch of oligonucleotides in the strand
X-B-Y-C (circled with a solid line) has a higher binding affinity
for B' than the "B" stretch of oligonucleotides in the strand
A-X-B, the equilibrium shifts in favor of the productive, strand
displacement reaction, which increases the probability or frequency
with which this reaction occurs. Both B's have the same
Watson-Crick base pairing sequence, but the interaction between
X-B-Y-C and the B' toe-hold is more favorable due to chemical
modifications.
[0044] Similarly, if certain stretches of nucleic acids are
replaced with analogs that disfavor binding, the equilibrium will
shift away from the clashes as well. For instance, if "B" in the
strand A-X-B (circled with a dashed line) has a lower binding for
B' than "B" in X-B-Y-C, then the equilibrium will shift away from
the toe-hold clash, improving the kinetics for a strand
displacement reaction.
[0045] Both approaches accelerate the speed at which a nucleic acid
network can process signals because both increase the residence
time of the desired strand on the toe-hold relative to the
residence time of the clashing strand or strands. This provides a
better chance that the strand displacement reaction will occur in a
given period of time and thus increases the rate at which the
network evaluates a given input or set of inputs. These
modifications have not previously been used to facilitate or
disfavor displacement reactions, to build significant DNA logic
gates and networks, or to propagate information.
[0046] There are many well-characterized nucleic acid modifications
that can be used in accordance with embodiments of the invention to
improve or reduce the thermodynamic properties of binding to
natural DNA or RNA. These include changes to the backbone, sugar,
or nucleobase of the oligonucleotide. These modifications also can
be used 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.
[0047] 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).
[0048] The general structures for certain analogs are shown in FIG.
5. These analogs all improve the thermodynamics of nucleic acid
hybridization reactions, allowing for tighter toe-hold binding and
therefore faster displacement. By using these analogs in specific
places of the logic network, the desired displacement reactions can
be strongly favored in comparison to the clashing interactions,
thus accelerating the rate of information processing.
[0049] 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 FIG.
6.
[0050] 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
(FIG. 7), all of which improve the binding affinity of a strand for
its reverse complement. Another opportunity that exists with
nucleobase modification involves pseudocomplementary bases. This
class of base analogs forms weak base pairs with one another but
forms strong base pairs with standard bases. One such pair of bases
is 2-aminoadenine (nA) and 2-thiothymine (sT).
[0051] These bases could be used to favor one strand binding while
disfavoring another, an example of increasing the likelihood of a
productive binding event while decreasing the likelihood of a clash
at the same time.
[0052] 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.
[0053] 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 METHODS IN
MOLECULAR BIOLOGY (Protocols for Oligonucleotides and Analogs) 1-17
(1993), in Reese, Colin B., Organic & Biomolecular Chemistry 3:
3851 (2005), and in Iyer, R. P.; and S. L. Beaucage, "7.05.
Oligonucleotide synthesis," 7 COMPREHENSIVE NATURAL PRODUCTS
CHEMISTRY (DNA and Aspects of Molecular Biology) 105-52 (1999), the
respective contents of which are hereby incorporated by reference
in their entirety.
[0054] If the backbone is being changed in a particular
modification, different chemistry will be employed. Such
modification chemistry is generally known 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,"
METHODS IN MOLECULAR BIOLOGY SERIES (Peptide Nucleic Acids), Humana
Press, http://www.springerlink.com/content/mr571738x7t65067/.
[0055] 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 more described in E. Uhlmann, et al., Angew. Chem. (Int'l ed.)
37: 2796-823 (1998), for example.
[0056] 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.
[0057] In accordance with some embodiments, the kinetics of the
chemically modified molecules for improved branch migration
reaction can be tested. One approach is to measure the improvement
that different modifications provide in accelerating or retarding
the branch migration reaction versus a natural polynucleotide using
a fluorescence kinetics assay (see FIG. 8, for example). Here, the
translator structure has a quencher on one strand and a fluorophore
on the other. In the absence of the displacement strand, the
fluorescence of the fluorophore is quenched, providing a baseline
for the reaction. When the displacement molecule, which could be
modified in the toe-hold and/or non-toe-hold regions, is added to
the translator, it will displace the strand with the quencher and
in doing so produce a fluorescent signal. Therefore, the rate of
fluorescence "turn-on" in the system indicates the kinetics of the
displacement reaction. This assay can be made more complex by
requiring a larger circuit to evaluate before a final,
fluorescence-producing displacement reaction or clashing strands
could be added to compete with the displacement strand for the
toe-hold.
[0058] The approaches in accordance with the invention also can
apply to any molecules, natural or artificial, that are suitable
for propagating information, so long as a first molecule and a
second molecule are involved, with the latter configured to bind
with the former. At least one of the first and second molecules,
respectively, has a chemical modification relative to a reference
molecule that has the same sequence content as the first molecule
or the second molecule, as the case may be. The modification causes
the binding to have a free energy different from that of a
reference binding between the reference molecule and the first or
second molecule, such that at least one of the following is
realized in the system: a probability of a productive binding is
raised; or a probability of an unproductive binding is reduced.
[0059] Pursuant to the invention, as indicated above, a multistep
process could be employed to achieve an improved nucleic acid
network. This would entail examining the full network of nucleic
acid translators under consideration, including any clashing
interactions, and then identifying the stretches of sequence that
need to be modified in order best to facilitate displacement events
and to minimize clashing.
[0060] With respect to any given strand displacement reaction,
these modifications could be made on the desired displacement
molecule, on the clashing molecule, or on both. This is so because,
for any translator or logic gate with an available toe-hold, there
will be a competition between the desired displacement molecule and
any clashing molecule to bind the toe-hold. Furthermore, increasing
the ability of the displacement molecule to bind the toe-hold
relative to the clashing molecule will increase the rate at which
the displacement reaction takes place. Thus, increasing the ability
of the displacement molecule to bind (lowering .DELTA.G of binding)
or decreasing the ability of the clashing molecule(s) to bind
(raising .DELTA.G of binding) will have the effect of increasing
the rate of the reaction. Since both of these changes in .DELTA.G
can be accomplished via chemical modifications, pursuant to the
invention, it is important when considering which stretches of
sequence to modify that one should explore the possibility of
modifying the displacement molecule alone, the clashing molecule
alone, and both of these together.
[0061] According to embodiments of the invention, two parameters
that could be varied in this analysis are (i) the rate constants
for the interaction between any two oligonucleotides and (ii) the
length of the toe-holds for all of the oligonucleotides in the
network. The former parameter would be dictated by the chemical
composition of the oligonucleotide and the .DELTA.G of its
interactions with other oligonucleotides. The latter parameter
would be the number of nucleotides in the toe-hold region of
sequence under consideration.
[0062] The kinetic rate constants needed to model a nucleic acid
network could be calculated from the .DELTA.G of hybridization
between any two oligonucleotides. For the natural bases and many of
the modifications, these values, which come from nearest-neighbor
parameters, have been reported in the literature. If they are not
known already, they can be experimentally determined through Van't
Hoff analysis of melting temperature data, differential scanning
calorimetry, or isothermal titration calorimetry. Each of these
methods would yield the thermodynamic parameters for the nucleic
acid interaction in question, which then can be used to determine
the .DELTA.G of the reaction at any temperature. The .DELTA.G value
then would be used to determine the equilibrium constant (K.sub.eq)
of the interaction. The equilibrium constant would be proportional
to the ratio of rate constants; hence, one could use known
constants to solve for the necessary kinetic rate constant for a
given reaction.
[0063] Thus, the full set of rate constants would be obtained for
the modifications under consideration for use in a nucleic acid
logic network, along with the lengths for all of the toe-holds.
With this information the system could be modeled to determine the
time course behavior of the network, and also to determine where
specific modifications could be employed to optimize the productive
displacement reactions relative to clashing interactions.
Additionally, the length of specific toe-hold regions could be
varied in the model. The network would be optimized by varying
modifications at specific locations and by simulating the reactions
in the network with different length toe-holds. These calculations
would be employed to determine the optimal modification and
toe-hold length for each component that produces the most favorable
time course behavior of the network.
[0064] The network also could be optimized by experimentally
changing the modifications and toe-hold lengths and then studying
the time course behavior in the laboratory, as opposed to
simulating the behavior with a mathematical model.
[0065] Given a fully optimized network, the necessary
oligonucleotides could be synthesized with standard phosphoramidite
chemistry or with the approach(es) reported in the literature for
specific modifications. Any translator or gate structure that
consists of more than one oligonucleotide could be constructed by
mixing the individual oligonucleotides together and then annealing
them, first by heating the mixture to above the melting temperature
of all possible duplexes and then slowly cooling it. This would
cause the oligonucleotides to hybridize, forming the most stable
structure that the particular set of nucleic acid sequences can
adopt. These structures then could be purified before they were
used in the network. Once all of the translators, gates, and other
components are designed, synthesized, and annealed, the network
could be applied to the target assay, diagnostic, or biological
system.
[0066] 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.
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