SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 21, 2018, is named P14626-02_SL.txt and is 18,621 bytes in size.

BACKGROUND OF THE INVENTION

A common design for smart materials and devices is a patterned architecture in which a specific chemical or physical stimulus produces swelling or shrinking of one or more material regions. Local, addressable control over this process can enable the multistate shape changes required for metamorphic materials or soft robots. Such control can be achieved by embedding wires that direct local mechanical deformations. However, wires add physical bulk and require batteries or tethering, limiting the scope of their operation.

Chemomechanically responsive materials present advantages in terms of autonomy, versatility, programmability, device integration and miniaturization as compared to materials with embedded wires. Chemicals can diffuse over large distances and into small or tortuous spaces, and the huge number of chemicals that can be synthesized offers unprecedented tunability and specificity.

Addressable control comparable to that in wired systems cannot be currently achieved by wire-free stimuli such as temperature, light, electromagnetic signals or pH. There is a commercial need to develop a wire-free responsive materials made of biomolecules

SUMMARY OF THE INVENTION

The inventor's focused on developing wire-free responsive materials beginning with building a library of biomolecules to achieve addressable control, such that each of a combinatorial variety of biomolecular species would direct the swelling of a specific material domain. Analogous to the way biomolecular stimuli would enable complex sensing, signal processing and feedback within biological cells and tissues.

One embodiment of the present invention is a programmable gel comprising: a first polymer comprising a first crosslink nucleic acid sequence; a second polymer comprising a second crosslink nucleic acid sequence; a first polymerizing hairpin capable of binding to the first crosslink nucleic acid sequence; a second polymerizing hairpin capable of binding to the second crosslink nucleic acid sequence; and a terminating hairpin capable of terminating polymerization. A suitable first crosslink nucleic acid sequence may comprise a first stem sequence, a first anchor B sequence, and a first anchor A sequence, for example. A suitable second crosslink nucleic acid sequence may comprise a second stem sequence that is complementary to the first stem sequence and a first dock A sequence; for example. A suitable first polymerizing hairpin may comprise the first stem sequence and the second stem sequence, a first dock B sequence, a second anchor A sequence that is complementary to the first anchor A sequence, and a second dock A sequence that is complementary to the first dock A sequence, for example. A suitable second polymerizing hairpin may comprise the first stem sequence, the second stem sequence, a second dock B sequence that is complementary to the first dock B sequence, the first dock A sequence; and the first anchor B sequence; for example. A suitable terminating hairpin may comprise the first stem sequence, the second stem sequence, the second dock A sequence, the second anchor A sequence and a terminating sequence. The programmable gel of claim2wherein the stem nucleic acid sequences comprise from 8 to 50 nucleic acids each, the dock A and dock B sequences comprise up to 10 nucleic acids each, and the anchor A and anchor B nucleic acid sequences comprise up to 10 nucleic acids each.

Another embodiment of the present invention is a method of making a programmable gel comprising: providing a gel comprising a first polymer comprising a first crosslink nucleic acid sequence; a second polymer comprising a second crosslink nucleic acid sequence wherein the first polymer and the second polymer are crosslinked by the first and second crosslink nucleic acid sequences; and adding a first polymerizing hairpin, a second polymerizing hairpin; and a terminating hairpin to the gel to produce a programmable gel; wherein a concentration ratio of the first polymerizing hairpin, the second polymerizing hairpin, and terminating hairpins are adjusted prior to addition to gel to control the timing and/or degree of swelling of the programmable gel.

Another embodiment of the present invention is an expandable gel comprising: a first polymer comprising a first crosslink nucleic acid sequence; a second polymer comprising a second crosslink nucleic acid sequence; a first polymerizing hairpin capable of binding to the first cross link nucleic acid sequence; and a second polymerizing hairpin capable of binding to the second crosslink nucleic acid sequence.

Another embodiment of the present invention is a method of making an expandable gel comprising: providing a gel comprising a first polymer comprising a first crosslink nucleic acid sequence; a second polymer comprising a second crosslink nucleic acid sequence that are crosslinked by the first and second crosslink nucleic acid sequences; adding a first polymerizing hairpin and a second polymerizing hairpin; and forming an expanded gel (until the hairpins are used up).

Another embodiment of the present invention is a photo patterning process to pattern gels into precisely-defined architectures comprising: providing a surface comprising a sacrificial layer; applying a collection of monomers, a photo-initiator, and a collection of crosslink nucleic acid sequences capable of binding to the monomers; placing a mask on top of the programmable gel wherein the mask is impenetrable by light and comprises one or more shapes wherein light is able to penetrate through the shapes on the mask; applying light to the mask so that light penetrates the shapes on the mask; polymerizing the monomers; binding the cross link nucleic acids to the monomers; forming a collection of polymers comprising attached nucleic acid sequences wherein the nucleic acid sequences crosslink the polymers and the collection of polymers comprises the one or more shapes; removing the mask; adding a ratio of polymerizing hairpins and terminating hairpins; and creating a gel having one or more precisely-defined architectures. A photopatterning process of the present invention may comprise the step of removing a collection of polymers from the surface prior to adding the ratio of polymerizing hairpins and terminating hairpins.

Any suitable polymer able to work with nucleic acid chemistry may be used in the present invention. Examples include water soluble polymers, polyisoprene, polystyrene, polypropylene, polyvinyl chloride, synthetic rubber, natural rubber, phenol formaldehyde resin (or Bakelite), neoprene, nylon, polyacrylonitrile, PVB, silicone, acrylamide, hydrogels, gelatin, alginate, collagen, cross-linked peptoids or nucleic acid polymers, agarose polysachrides, hyaluronic acid acrylate, cross-linked acrylate, methacrylate, and acrylic acid, as examples.

One embodiment of the present invention are methods of making programmable gels. The methods of the present invention include providing a gel comprising a first polymer comprising a tag. Adding a first polymerizing hairpin, a second polymerizing hairpin, and a terminating hairpin to the gel. Producing a programmable gel that is expanding compared to a reference gel wherein the first polymerizing hairpin, the second polymerizing hairpin and a terminating hairpin have not been added. The gels of the present invention are programmable because the concentration ratio of starting materials are predetermined prior to their addition to a gel so as to control the timing and/or degree of swelling of a programmable gel. For example, a first polymerizing hairpin, a second polymerizing hairpin, and terminating hairpins are adjusted prior to addition to a gel to control the timing and/or degree of swelling of the programmable gel. The expansion and/or the contraction of gels of the present invention occurs by annealing nucleic acid sequences of the present invention. For example a tag is a single stranded nucleic acid such as an RNA or DNA that may include a dock B sequence and a tag anchor A sequence. The first polymerizing hairpin comprises a first stem sequence and a second stem sequence, a first dock B sequence, a second anchor A sequence that is complementary to the first anchor A sequence and a second dock A sequence that is complementary to a first dock A sequence. The first dock B sequence binds to the tag dock B sequence and where the second anchor A sequence binds to the tag anchor A sequence. The second polymerizing hairpin comprises the first stem sequence, the second stem sequence, a second dock B sequence that is complementary to the first dock B sequence, the first dock A sequence; and a first anchor B sequence. The terminating hairpin comprises the first stem sequence, the second stem sequence, the second dock A sequence, the second anchor A sequence and a terminating sequence.

Another embodiment of the present invention are methods of making a programmable gel that expands and contracts. The methods include providing a gel comprising a first polymer comprising a first crosslink nucleic acid sequence and a second polymer comprising a second crosslink nucleic acid sequence. Crosslinking the first polymer and the second polymer by the first and second crosslink nucleic acid sequences and a third crosslinker. Adding a first polymerizing hairpin, a second polymerizing hairpin; and a terminating hairpin to the gel. Producing a programmable gel that expands. Adding a first reversal strand, a second reversal strand to the programmable gel and contracting the gel compared to a reference gel that does not have a first and second reversal strand added. The methods may include a third polymerizing hairpin and fourth polymerizing hairpin are added to the contracted gel and expands the contracted gel forming an expanded contracted gel. The methods may include adding a third reversal strand and a fourth reversal strand to the expanded contracted gel and contracting the expanded, contracted gel. A programmable gel of the present invention may go through many cycles of expansion, contraction, or a combination thereof. Each expansion cycle may use the same polymerizing hairpins as the previous cycle, different polymerizing hairpins, or a combination thereof. Each contraction cycle may use the same reversal strands as the previous cycle, different reversal strands, or a combination thereof. In some methods, a gel is programmable because a concentration ratio of the first polymerizing hairpin, the second polymerizing hairpin, the terminating hairpins, the first reversal strand, and the second reversal strand are adjusted prior to addition to a gel to control the timing and/or degree of swelling and/or contraction of the gel. A suitable third crosslink comprises a physical, ionic, chemical, or nucleic acid crosslink. In some methods of the present invention the first and second polymerizing hairpins, terminating hairpins, and first and second reversal strands are incapable of interacting with the third crosslinker.

As mentioned, the expansion and/or contraction of gels of the present invention occurs by annealing nucleic acid sequences of the present invention. For example, the first and the second reversal strands of the present invention are nucleic acids such as RNA or DNA. In some methods of the present invention the first reversal strand interacts with the first polymerizing hairpin and the second reversal strand interacts with the second polymerizing hairpin.

Another embodiment of the present invention are methods of gel contraction. The methods include providing a gel comprising a first polymer comprising a first crosslink nucleic acid sequence and a second polymer. The second polymer comprises a second crosslink nucleic acid sequence and the first polymer and the second polymer are crosslinked by the first and second crosslink nucleic acid sequences. The gel also includes a third crosslinker, a first polymerizing hairpin, a second polymerizing hairpin, and a terminating hairpin. To this gel is added a first and a second reversal strand that causes the gel to contract when compared to a reference gel that does not include the first and second reversal strand.

FIGS. 1, 25, and 26are color coded so that the gray region is an example of the stem sequence; the pink region is an example of the dock A sequence; the cyan region is an example of the anchor A sequence; the green region is an example of the anchor B sequence; and the purple is an example of the dock B sequence.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

“Crosslinked nucleic acid sequence” means a double stranded nucleic acid sequence that typically contains anchor, stem, and dock sequences.

“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

A “reference sequence” is a defined sequence used as a basis for sequence comparison.

By “specifically binds” is meant a nucleic acid that recognizes and binds a second complementary nucleic acid of the invention, but which does not substantially recognize and bind other non-complementary nucleic acid molecules in a sample.

“Tag” means a single stranded nucleic acid sequence that typically contains a dock and stem sequences.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By “substantially identical” is meant a nucleic acid molecule exhibiting at least 50% identity to a reference nucleic acid sequence. Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the nucleic acid sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3and e−100indicating a closely related sequence.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

