Compositions, systems, and methods for data storage by modifying and reading clusters of transformable monomers within polymers - Patents.com

Abstract

Data-encodeable polymers, including nucleic acid polymers, for data storage and related methods are described. In general, a nucleic acid polymer may have chemically modifiable structures that are repeated repeatedly along the polymer. The chemically modifiable structures can be modified to encode data. A data encoding device may have the spatial resolution to modify a series of contiguous chemically modifiable structures resulting in clusters of modified structures. A variety of methods can be utilized to generate data-encodeable nucleic acid polymers. A variety of methods can be utilized to encode data-encodeable nucleic acid polymers. A variety of decoding methods can be utilized to decode the encoded nucleic acid or other polymers.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 268,354, filed February 22, 2022, and U.S. Provisional Patent Application No. 63 / 374,733, filed September 6, 2022, both of which are hereby incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety, said XML copy having a creation date of Feb. 20, 2023, named NAIO_706.601_SL.xml, is 9,568 bytes in size, and is 12,288 bytes in size on disk. [Background technology]

[0003] background The use of polymer molecules to encode digital data is under very active research. In principle, sequences of repeating spaced functional groups attached to monomers or selected monomers can be encoded into ones and zeros (bits) in the binary sense. Such molecular data encoding offers the potential for very high data density, since many bits of data can be encoded in a single molecule. Furthermore, some polymers (such as DNA) can remain stable longer than electronic or magnetic media, thus eliminating the need to frequently rewrite data for archival storage.

[0004] Because DNA is easy to synthesize and analyze, this biopolymer has attracted much more attention for molecular data encoding research. Solid-phase synthesis allows DNA to be assembled into desired and / or arbitrary sequences that can be encoded into a string of binary data. Furthermore, sequencing methods are rapidly becoming more efficient and inexpensive, allowing data to be "read" and therefore encoded. However, the stepwise assembly of DNA by solid-phase synthesis limits the length of the polymer to about 200 monomers, thereby severely limiting the amount of data that can be encoded within a single molecule. While it would be ideal to encode data into very long DNA polymers (e.g., greater than 10 kilobases), methods for creating very long DNA of arbitrary sequence are limited, slow, or nonexistent. Biologically synthesized DNA, while very long, does not exist in arbitrary sequence, limiting the ability to efficiently encode data into these polymers. Summary of the Invention [Means for solving the problem]

[0005] overview When using single dye imaging to read and write data stored in biological polymers, single dye imaging, for example, when considering a single chromophore molecule attached to a single nucleobase of a DNA polymer, may require highly specialized and sensitive cameras due to the very weak fluorescence emitted by a single chromophore. Localizing the position of the dye in the image relative to other dyes that may be obscured by diffraction may require computer analysis. Furthermore, when using a single photomodifiable group as a bit, one bit must be separated from adjacent bits because the loss of one bit will cause an error in the series of bits. Furthermore, shining light on a single dye and modulating it may cause some stochastic fluctuations, which may cause errors including not modifying the chromophore when writing is intended. For example, the method may include removing the dye from a linker attached to the DNA backbone by light to encode digital information, and knowledge and manipulation of the position of the dye relative to the secondary structure of the DNA may be required in a nanopore sequencer. Thus, if the method involves nanopore sequencing, highly specialized and sensitive cameras and computer analysis may be required to localize the position of the dye in the image relative to other dyes that may be obscured by diffraction, as well as forward and reverse currents to move the DNA back and forth to process the information at the single nucleotide level, in addition to errors arising from stochastic fluctuations, all of which pose barriers to the adoption of such improved data storage technologies.

[0006] In response to such barriers in the art, methods are provided herein for the use of chemically modifiable structures (e.g., dye clusters in DNA or RNA) incorporated into biological polymers such as DNA or RNA to encode bits, where the clusters comprise a size larger than would be obscured by diffraction or other optical aberrations. Such embodiments may allow data encoded in the polymer to be read without the use of single molecule imaging methods, allowing the information encoded therein to be read using standard microscope cameras or detectors. In some embodiments, methods and compositions are provided herein that include clusters that may allow data to be written and read faster and at less cost.

[0007] Aspects disclosed herein provide a method of encoding data on a writeable polymer, comprising: providing a data-encodeable nucleic acid polymer having a sequence of nucleotides in which one or more chemically-modifiable structures are repeatedly repeated, each of the one or more chemically-modifiable structures being attached to the nucleic acid polymer, the one or more chemically-modifiable structures being capable of being modified to a second structural state by a pulse of light or redox energy; and utilizing a data encoding device to selectively modify a subset of the one or more chemically-modifiable structures along the nucleic acid polymer to the second structural state, resulting in a data-encoded polymer defined by a plurality of modified structures, the modified structures being arranged as clusters of repeatedly repeating units, the length of each cluster being determined by the spatial resolution of the data encoding device. In some embodiments, the cluster comprises at least two consecutively modified structures. In some embodiments, the cluster comprises at least four consecutively modified structures. In some embodiments, the cluster comprises at least 21 consecutively modified structures. In some embodiments, the selectively modifying step comprises forming a single cluster at a time. In some embodiments, the selectively modifying step comprises simultaneously modifying a plurality of chemically-modifiable structures to form a cluster. In some embodiments, the selectively modifying step comprises modifying a first subset of the one or more chemically-modifiable structures along the nucleic acid polymer to form a first cluster, not modifying a second subset of the one or more chemically-modifiable structures along the nucleic acid polymer that are positioned after the first cluster in the 5' to 3' direction, and then modifying a third subset of the one or more chemically-modifiable structures along the nucleic acid polymer to form a third cluster that are positioned along the nucleic acid polymer that are positioned after the second subset of the one or more chemically-modifiable structures in the 5' to 3' direction.In some embodiments, the cluster of repeating units comprises consecutive repeating units. In some embodiments, the cluster of consecutive repeating units comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, or 21 repeating units of the modified structure. In some embodiments, the cluster of consecutive repeating units comprises at least 21 repeating units. In some embodiments, the cluster of consecutive repeating units comprises at least 625 repeating units. In some embodiments, the cluster of consecutive repeating units comprises at least about 0.6 kbp of repeating units. In some embodiments, the cluster of consecutive repeating units comprises at least about 1.2 kbp of repeating units. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 21, 24, 28, 32, 40, 80, 120 or 625 nucleotides. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 21 units. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 625 units. In some embodiments, the cluster of repeating units comprises at least about 0.6 kbp of repeating units. In some embodiments, the cluster of repeating units comprises at least about 1.2 kbp of repeating units. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance on the order of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance on the order of at least 0.6 microns over the length of the polymer.In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance on the order of at least 1 micron for the length of the polymer. In some embodiments, each chemically modifiable structure of the one or more chemically modifiable structures includes one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore. In some embodiments, the data encoding device includes a nanopore, and the method further includes passing the encodable nucleic acid polymer through the nanopore of the data encoding device, the nanopore including a means for applying a pulse of light or redox energy to a subset of the one or more chemically modifiable structures to a second structural state. In some embodiments, each chemically modifiable structure of the one or more chemically modifiable structures includes a caging group, a quencher, a photobleachable fluorophore, a photoconvertible fluorophore, or any combination thereof. In some embodiments, a first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a first wavelength of light. In some embodiments, the second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that can be photobleached by a pulse of light energy at a second wavelength of light. In some embodiments, the first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that cannot be photobleached by a pulse of light energy at a second wavelength of light, and the second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that cannot be photobleached by a pulse of light energy at a first wavelength of light. In some embodiments, the first chemically modifiable structure and the second chemically modifiable structure are attached to a repeated nucleotide structure. In some embodiments, the nucleic acid polymer is double-stranded, and the first chemically modifiable structure is attached to a repeated nucleotide structure on the top strand of the nucleic acid polymer, and the second chemically modifiable structure is attached to a repeated nucleotide structure on the bottom strand of the nucleic acid polymer.In some embodiments, the one or more chemically modifiable structures are repeatedly repeated at every second nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth nucleotide, every ninth nucleotide, every tenth nucleotide, every eleventh nucleotide, every twelfth nucleotide, every thirteenth nucleotide, every fourteenth nucleotide, every fifteenth nucleotide, every sixteenth nucleotide, every seventeenth nucleotide, every eighteenth nucleotide, every nineteenth nucleotide, every twentieth nucleotide, every twenty-first nucleotide, every twenty-second nucleotide, every twenty-third nucleotide, every twenty-fourth nucleotide, or every twenty-fifth nucleotide, as follows: In some embodiments, the polymer comprises DNA, RNA, phosphorothioate DNA, glycerol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro-DNA 2'-O-methyl RNA, or locked nucleic acid (LNA). In some embodiments, the method further comprises decoding the encoded data of the selectively modified nucleic acid polymer. In some embodiments, the encoded data is decoded by passing the selectively modified nucleic acid polymer through a nanopore of a data decoding device, the nanopore of the data decoding device including a means for detecting the chemically modified structures in each cluster. In some embodiments, the data decoding device is the same device as the data encoding device. In some embodiments, the nanopore for decoding the data is the same nanopore for encoding the data. In some embodiments, the encoded data is decoded by stretching and imaging the selectively modified nucleic acid polymer. In some embodiments, the photo-modifiable group includes a photo-removable leaving group that is not attached by a linker. In some embodiments, the photo-modifiable group includes a photo-removable leaving group that is cleavable by light at a carbonyl bond, a thio (S) bond, or an NR2 bond. In some embodiments, the plurality of convertible nucleobases are converted from a first state to a second state by light at a wavelength of 325 nm, 360 nm, or 400 nm.In some embodiments, the plurality of convertible nucleobases are converted from a first state to a second state by light of wavelengths between 400 nm and 850 nm. In some embodiments, at least three convertible residues in the first state are between data encoding segments of the polymer comprising the plurality of convertible residues in the second state. In some embodiments, at least three convertible residues in the first state are utilized for every nanometer of spatial resolution at which data is encoded. In some embodiments, the method further comprises a plurality of spacers between data encoding segments of the polymer. In some embodiments, at least three spacer residues are utilized for every nanometer of spatial resolution at which data is encoded. In some embodiments, the spacer residues are nucleobases that are not photomodifiable. In some embodiments, the convertible residues comprise covalently attached monomeric units. In some embodiments, the chemically modifiable structure is convertible comprising a photomodifiable group that comprises a leaving group that is photoremovable. In some embodiments, the convertible residues comprise covalently attached monomeric units that are linked in series along substantially the entire length of the polymer. In some embodiments, the convertible residues comprise covalently linked monomeric units and data is encoded in the covalently attached monomeric units. In some embodiments, the convertible residues comprise covalently linked monomeric units and data is encoded in consecutive covalently attached monomeric units. In some embodiments, the polymer is defined by covalently linked monomeric units and does not hybridize in a side-by-side orientation. In some embodiments, the polymer is defined by covalently linked monomeric units and does not hybridize in a side-by-side orientation with a second polymer to which the polymer is not covalently linked. In some embodiments, the method further comprises forming a single-stranded polymer. In some embodiments, the writeable polymer is DNA and the method further comprises creating a second polymer that is complementary to the data-encoded polymer over approximately at least a portion of the data-encoded polymer.In some embodiments, the method further comprises hybridizing at least a portion of the data-encoded polymer to a second polymer. In some embodiments, the method further comprises hybridizing at least a portion of the data-encoded polymer to a second polymer in a 5' to 3' direction. In some embodiments, the polymer comprises up to two consecutive covalently linked polymer molecules. In some embodiments, the two consecutive covalently linked polymer molecules form a DNA duplex. In some embodiments, the DNA duplex is hybridized in a 3' to 5' direction. In some embodiments, the repeat interval is. The spaced convertible residues are located on a single covalently linked polymer. In some embodiments, the method further comprises heating, cooling, reheating, or any combination thereof, the data-encoded polymer, wherein the data-encoded polymer maintains its residue position in approximately the 3' to 5' direction during the heating, cooling, reheating, or any combination thereof. In some embodiments, the chemically modifiable moieties are directly linked to one or more nucleobases. In some embodiments, the chemically modifiable moieties are spaced apart in a repeating manner approximately along the length of the polymer. In some embodiments, the spaced apart repeating manner along the length of the polymer is present at least every 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 2500, or at least 5000 spacer residues. In some embodiments, the selectively modifying step comprises removing a photoremovable leaving group at least every 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 2500, or 5000 spacer residues. In some embodiments, the selectively modifying step comprises locally photocleaving a single photoremovable leaving group at a time. In some embodiments, there is no secondary structure attached to the polymer other than the chemically modifiable moiety directly linked to one or more nucleobases. In some embodiments, the polymer does not have any data encoded thereon when the plurality of convertible residues are in a first state. In some embodiments, the polymer is a writeable polymer configured to write data onto the polymer. In some embodiments, the polymer encodes data when at least a portion of the plurality of convertible residues are in a second state.In some embodiments, the selectively modifying step encodes data by cleaving at least a portion of the photoremovable leaving group from the chemically-modifiable moiety to form a data-encoded polymer. In some embodiments, the selectively modifying step includes reagent-free cleavage of the photoremovable leaving group from the nucleobase of one or more nucleobases. In some embodiments, the selectively modifying step includes translocating the polymer through the nanopore. In some embodiments, the selectively modifying step includes translocating the polymer in one direction through the nanopore without translocating the polymer in the reverse direction through the nanopore. In some embodiments, the selectively modifying step includes translocating the polymer through the nanopore at a continuous speed. In some embodiments, the selectively modifying step includes writing data along the length of the polymer over a length of at least 1000, 2000, 3000, 4000, 5000, 10000, or 500000 residues. In some embodiments, the selectively modifying step does not include oxidizing one or more nucleobases. In some embodiments, the chemically modifiable moiety comprises a molecule comprising a plurality of distinct atoms. In some embodiments, the chemically modifiable moiety comprises a molecule comprising an atom other than oxygen (O). In some embodiments, the photoremovable leaving group comprises a molecule comprising a plurality of distinct atoms. In some embodiments, the photoremovable leaving group comprises a molecule comprising an atom other than O. In some embodiments, the method further comprises reading the data encoded on the polymer by passing the data encoded writeable polymer through a data reading device to identify whether each of the plurality of transformable residues that pass through the data reading device comprises a photoremovable leaving group.

[0008] Aspects disclosed herein provide polymers encoded with data using data encoding with a resolution that results in clusters of modified structures spaced apart in a repeating manner, the polymer comprising a nucleic acid polymer having a sequence of nucleotides in which one or more chemically modifiable structures are repeatedly repeated, the nucleic acid polymer comprising clusters of repeating units of the modified structures, the clusters being repeatedly repeated along the nucleic acid, the modified structures being chemically modified from a first structural state to a second structural state by a pulse of light or redox energy, the plurality of clusters defining encoded data determined by the modified structures within each cluster. In some embodiments, the clusters of repeating units comprise contiguous repeating units of the modified structures in the second structural state. In some embodiments, the clusters of contiguous repeating units comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units of the modified structures. In some embodiments, the clusters of contiguous repeating units comprise at least 21 repeating units of the modified structures. In some embodiments, the clusters of contiguous repeating units comprise at least 625 repeating units of the modified structures. In some embodiments, the cluster of consecutive repeating units comprises at least about 0.6 kbp of repeating units of the modified structure. In some embodiments, the cluster of consecutive repeating units comprises at least about 1.2 kbp of repeating units of the modified structure. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 1 micron over the length of the polymer. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 2, 3, 4, 5, 6, 7, 8, 9 or 10 units.In some embodiments, the cluster of repeating units is repeated at least every 21 units. In some embodiments, the cluster of repeating units is repeated at least every 625 units. In some embodiments, the cluster of repeating units comprises at least about 0.6 kbp of repeating units. In some embodiments, the cluster of repeating units comprises at least about 1.2 kbp of repeating units. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least about 1 micron over the length of the polymer. In some embodiments, the chemically modifiable structure comprises a photomodifiable group comprising a photoremovable leaving group. In some embodiments, the photomodifiable group comprises a photoremovable leaving group that is not attached by a linker. In some embodiments, the photo-modifiable group comprises a photoremovable leaving group cleavable by light at a carbonyl bond, a thio (S) bond, or an NR2 bond. In some embodiments, the plurality of convertible nucleobases are converted from a first state to a second state by light at a wavelength of 325 nm, 360 nm, or 400 nm. In some embodiments, the plurality of convertible nucleobases are converted from a first state to a second state by light at a wavelength between 400 nm and 850 nm. In some embodiments, at least three convertible residues in the first state are between data encoding segments of the polymer comprising a plurality of convertible residues in the second state. In some embodiments, at least three convertible residues in the first state are utilized for every nanometer of spatial resolution at which data is encoded. In some embodiments, the polymer further comprises a plurality of spacers between data encoding segments of the polymer. In some embodiments, at least three spacer residues are utilized for every nanometer of spatial resolution at which data is encoded.In some embodiments, the spacer residue is a nucleobase that is not photomodifiable. In some embodiments, the convertible residue comprises a covalently attached monomeric unit. In some embodiments, the convertible residue comprises covalently linked monomeric units that are consecutively linked over substantially the entire length of the polymer. In some embodiments, the convertible residue comprises covalently linked monomeric units, and data is encoded in the covalently linked monomeric units. In some embodiments, the convertible residue comprises covalently linked monomeric units, and data is encoded in the consecutive covalently linked monomeric units. In some embodiments, the polymer is defined by covalently linked monomeric units and does not hybridize in a side-by-side orientation. In some embodiments, the polymer is defined by covalently linked monomeric units, and does not hybridize in a side-by-side orientation with a second polymer to which the polymer is not covalently linked. In some embodiments, the polymer comprises a single molecule that forms a single-stranded polymer. In some embodiments, the polymer is defined by up to two DNA polymer molecules. In some embodiments, the polymer is defined by up to two hybridized DNA polymer molecules. In some embodiments, the polymer is defined by up to two hybridized DNA polymer molecules hybridized in a 5' to 3' direction. In some embodiments, the polymer comprises up to two consecutive covalently linked polymer molecules. In some embodiments, the two consecutive covalently linked polymer molecules form a DNA duplex. In some embodiments, the DNA duplex is hybridized in a 3' to 5' direction. In some embodiments, the repeatedly spaced convertible residues are located on a single covalently linked polymer. In some embodiments, the polymer maintains its residue position in approximately a 3' to 5' direction when heated, cooled, reheated, or any combination thereof. In some embodiments, the polymer is not linked to any other polymer by hybridization. In some embodiments, the polymer comprises one or more nucleobases, and the chemically modifiable moiety is directly linked to one or more nucleobases.In some embodiments, the convertible residues comprising the chemically modifiable moiety are spaced apart in a repeating manner approximately along the length of the polymer. In some embodiments, the repeating spacing along the length of the polymer occurs at least every 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 2500, or at least 5000 spacer residues. In some embodiments, the polymer is configured for localized photocleavage of a single removable leaving group at a time. In some embodiments, there is no secondary structure associated with the polymer other than the chemically modifiable moiety directly linked to one or more nucleobases. In some embodiments, the polymer does not have any data encoded thereon when the plurality of convertible residues are in a first state. In some embodiments, the polymer is a writeable polymer configured to write data onto the polymer. In some embodiments, the polymer is encoded with data when at least a portion of the plurality of convertible residues are in a second state. In some embodiments, the polymer is configured to encode data by cleaving at least a portion of the photoremovable leaving group from the polymer. In some embodiments, the chemically modifiable moiety is configured to be cleaved by one or more nucleobases without the use of chemical reagents. In some embodiments, the polymer is configured to translocate unidirectionally through the nanopore. In some embodiments, the polymer is configured to translocate unidirectionally through the nanopore without translocating in the reverse direction through the nanopore. In some embodiments, the polymer is at least 1000, 2000, 3000, 4000, 5000, 10000, or 500000 residues in length. In some embodiments, the polymer in the second state does not include oxidized nucleobases. In some embodiments, the second state is a written form that does not include oxidized nucleobases. In some embodiments, the chemically modifiable moiety includes a chemical residue that includes a plurality of atoms.In some embodiments, the polymer in the second state does not include a chemically modifiable moiety. In some embodiments, the polymer in the second state does not include a chemical moiety comprising a plurality of atoms. In some embodiments, the chemically modifiable moiety comprises a molecule comprising an atom other than O (oxygen). In some embodiments, the photoremovable leaving group comprises a molecule comprising a plurality of atoms. In some embodiments, the photoremovable leaving group comprises a molecule comprising a plurality of distinct atoms. In some embodiments, the photoremovable leaving group comprises a molecule comprising an atom other than O. In some embodiments, the first structural state of each modified structure of the plurality of modified structures comprises one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore. In some embodiments, the modified structures of the first cluster are one or more photobleached fluorophores that have been photobleached by a pulse of light energy at a first wavelength of light. In some embodiments, the modified structures of the second cluster are one or more photobleached fluorophores that have been photobleached by a pulse of light energy at a second wavelength of light. In some embodiments, the first cluster further comprises one or more photobleachable fluorophores that are photobleachable at a second wavelength of light but are not photobleached, and the second cluster further comprises one or more photobleachable fluorophores that are photobleachable at a first wavelength of light but are not photobleached. In some embodiments, each photobleachable fluorophore that is photobleachable at a first wavelength in the first cluster has been photobleached by a pulse of light energy at the first wavelength of light, and each photobleachable fluorophore that is photobleachable at a second wavelength in the second cluster has been photobleached by a pulse of light energy at the second wavelength of light. In some embodiments, the modified structure and one or more photobleachable fluorophores of the first cluster are attached to a repeated nucleotide structure. In some embodiments, the nucleic acid is double-stranded, and the modified structure of the first cluster is attached to a repeated nucleotide structure on the top strand of the nucleic acid polymer, and the one or more photobleachable fluorophores of the first cluster are attached to a repeated nucleotide structure on the bottom strand of the nucleic acid polymer.In some embodiments, the length of each cluster of the plurality of clusters is equal to the spatial resolution of the pulse of light or redox energy used to modify the plurality of chemically modified structures from a first structural state to a second structural state. In some embodiments, the nucleic acid polymer comprises a plurality of intervals between each cluster of the plurality of clusters. In some embodiments, each interval of the plurality of intervals is modified by a pulse of light or redox energy. The polymer comprises one or more unmodified chemically modifiable structures. In some embodiments, the one or more modified structures are repeatedly repeated at every second nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth nucleotide, every ninth nucleotide, every tenth nucleotide, every eleventh nucleotide, every twelfth nucleotide, every thirteenth nucleotide, every fourteenth nucleotide, every fifteenth nucleotide, every sixteenth nucleotide, every seventeenth nucleotide, every eighteenth nucleotide, every nineteenth nucleotide, every twentieth nucleotide, every twenty-first nucleotide, every twenty-second nucleotide, every twenty-third nucleotide, every twenty-fourth nucleotide, or every twenty-fifth nucleotide, as follows: In some embodiments, the polymer comprises DNA, RNA, phosphorothioate DNA, glycerol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro-DNA 2'-O-methyl RNA, or locked nucleic acid (LNA).

[0009] Aspects disclosed herein provide a data-encodeable polymer for data encoding utilizing repeatedly spaced chemically modifiable structures, comprising a nucleic acid polymer having a sequence of nucleotides in which one or more chemically modifiable structures are repeatedly repeated, each of the one or more chemically modifiable structures being attached onto the nucleic acid polymer, and the one or more chemically modifiable structures are capable of being modified from a first structural state to a second structural state in a cluster of a plurality of chemically modified structures by a pulse of light or redox energy. In some embodiments, the cluster of repeating units comprises contiguous repeating units. In some embodiments, the cluster of contiguous repeating units comprises at least 4, 8, 10, 12, 16, or 20 repeating units. In some embodiments, the cluster of contiguous repeating units comprises at least 21 repeating units. In some embodiments, the cluster of contiguous repeating units comprises at least 625 repeating units. In some embodiments, the cluster of contiguous repeating units comprises at least about 0.6 kbp of repeating units. In some embodiments, the cluster of consecutive repeating units comprises at least about 1.2 kbp of repeating units. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 1 micron or so over the length of the polymer. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 10 units. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 21 units. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 625 units.In some embodiments, the cluster of repeating units comprises at least about 0.6 kbp of repeating units. In some embodiments, the cluster of repeating units comprises at least about 1.2 kbp of repeating units. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least about 1 micron over the length of the polymer. In some embodiments, each chemically modifiable structure of the one or more chemically modifiable structures comprises one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore. In some embodiments, a first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a first wavelength of light. In some embodiments, the first chemically modifiable structure is attached to a repeated nucleotide structure. In some embodiments, the second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that can be photobleached by a pulse of light energy at a second wavelength of light. In some embodiments, the first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that cannot be photobleached by a pulse of light energy at a second wavelength of light, and the second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that cannot be photobleached by a pulse of light energy at a first wavelength of light. In some embodiments, the first chemically modifiable structure and the second chemically modifiable structure are attached to a repeated nucleotide structure. In some embodiments, the nucleic acid polymer is double-stranded, and the first chemically modifiable structure is attached to a repeated nucleotide structure on the top strand of the nucleic acid polymer, and the second chemically modifiable structure is attached to a repeated nucleotide structure on the bottom strand of the nucleic acid polymer.In some embodiments, the one or more chemically modifiable structures comprise a photoconvertible fluorophore that exists in a first structural state having a first emission wavelength and is convertible to a second structural state having a second emission wavelength by a pulse of light or redox energy. In some embodiments, the conversion of the photoconvertible fluorophore from the first structural state to the second structural state is by a pulse of light at a first wavelength, and the photoconvertible fluorophore is convertible to a third structural state having a third emission wavelength by a pulse of light at a second wavelength. In some embodiments, one or more chemically modifiable structures are repeatedly repeated at every second nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth nucleotide, every ninth nucleotide, every tenth nucleotide, every eleventh nucleotide, every twelfth nucleotide, every thirteenth nucleotide, every fourteenth nucleotide, every fifteenth nucleotide, every sixteenth nucleotide, every seventeenth nucleotide, every eighteenth nucleotide, every nineteenth nucleotide, every twentieth nucleotide, every twenty-first nucleotide, every twenty-second nucleotide, every twenty-third nucleotide, every twenty-fourth nucleotide, or every twenty-fifth nucleotide, as follows: In some embodiments, the chemically modifiable structure comprises a photomodifiable group that comprises a photoremovable leaving group. In some embodiments, the chemically modifiable structure comprises a photomodifiable group that comprises a photoremovable leaving group. In some embodiments, the photomodifiable group comprises a photoremovable leaving group that is not linked by a linker. In some embodiments, the photomodifiable group comprises a photoremovable leaving group that is cleavable by light at a carbonyl bond, a thio (S) bond, or an NR2 bond. In some embodiments, the plurality of convertible nucleobases are converted from a first state to a second state by light at a wavelength of 325 nm, 360 nm, or 400 nm.In some embodiments, the plurality of convertible nucleobases are converted from a first state to a second state by light of a wavelength between 400 nm and 850 nm. In some embodiments, at least three convertible residues in the first state are between data encoding segments of the polymer comprising a plurality of convertible residues in the second state. In some embodiments, at least three convertible residues in the first state are utilized for every nanometer of spatial resolution at which data is encoded. In some embodiments, the polymer further comprises a plurality of spacers between data encoding segments of the polymer. In some embodiments, at least three spacer residues are utilized for every nanometer of spatial resolution at which data is encoded. In some embodiments, the spacer residues are nucleobases that are not modifiable by light. In some embodiments, the convertible residues comprise covalently attached monomeric units. In some embodiments, the convertible residues comprise covalently linked monomeric units that are linked in sequence over substantially the entire length of the polymer. In some embodiments, the convertible residues comprise covalently linked monomeric units and data is encoded in the covalently linked monomeric units. In some embodiments, the convertible residues comprise covalently linked monomeric units and data is encoded in consecutive covalently linked monomeric units. In some embodiments, the polymer is defined by covalently linked monomeric units and does not hybridize in a side-by-side orientation. In some embodiments, the polymer is defined by covalently linked monomeric units and does not hybridize in a side-by-side orientation with a second polymer to which the polymer is not covalently linked. In some embodiments, the polymer comprises a single molecule that forms a single stranded polymer. In some embodiments, the polymer is defined by up to two DNA polymer molecules. In some embodiments, the polymer is defined by up to two hybridized DNA polymer molecules. In some embodiments, the polymer is defined by up to two hybridized DNA polymer molecules hybridized in a 5' to 3' orientation. In some embodiments, the polymer comprises up to two consecutive covalently linked polymer molecules.In some embodiments, two consecutive covalently linked polymer molecules form a DNA duplex. In some embodiments, the DNA duplex is hybridized in a 3' to 5' direction. In some embodiments, the repeatedly spaced convertible residues are located on a single covalently linked polymer. In some embodiments, the polymer maintains its residue location in approximately a 3' to 5' direction when heated, cooled, reheated, or any combination thereof. In some embodiments, the polymer is not linked to any other polymer by hybridization. In some embodiments, the polymer comprises one or more nucleobases, and the chemically modifiable moiety is directly linked to one or more nucleobases. In some embodiments, the convertible residues comprising the chemically modifiable moiety are repeatedly spaced approximately along the length of the polymer. In some embodiments, the repeat spacing along the length of the polymer occurs at least every 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 2500, or at least 5000 spacer residues. In some embodiments, the polymer is configured for localized photocleavage of a single removable leaving group at a time. In some embodiments, there is no secondary structure attached to the polymer other than the chemically modifiable moiety directly linked to one or more nucleobases. In some embodiments, the polymer does not have any data encoded thereon when the plurality of convertible residues are in a first state. In some embodiments, the polymer is a writeable polymer configured to write data onto the polymer. In some embodiments, the polymer is encoded with data when at least a portion of the plurality of convertible residues are in a second state. In some embodiments, the polymer is configured to encode data by cleaving at least a portion of the photo-removable leaving groups from the polymer.In some embodiments, the chemically modifiable moiety is configured to be cleaved by one or more nucleobases without the use of chemical reagents. In some embodiments, the polymer is configured to translocate unidirectionally through the nanopore. In some embodiments, the polymer is configured to translocate unidirectionally through the nanopore without translocating in the reverse direction through the nanopore. In some embodiments, the polymer is at least 1000, 2000, 3000, 4000, 5000, 10000, or 500000 residues in length. In some embodiments, the polymer in the second state is , does not include an oxidized nucleobase. In some embodiments, the second state is a written form that does not include an oxidized nucleobase. In some embodiments, the chemically modifiable moiety includes a chemical residue that includes multiple atoms. In some embodiments, the polymer in the second state does not include a chemically modifiable moiety. In some embodiments, the polymer in the second state does not include a chemical residue that includes multiple atoms. In some embodiments, the chemically modifiable moiety includes a molecule that includes an atom other than O (oxygen). In some embodiments, the polymer of claim 193, wherein the photoremovable leaving group includes a molecule that includes multiple atoms. In some embodiments, the photoremovable leaving group includes a molecule that includes multiple distinct atoms. In some embodiments, the photoremovable leaving group includes a molecule that includes an atom other than O. In some embodiments, the polymer is configured to encode data in a cluster of chemically modified structures converted from the chemically modifiable structure. In some embodiments, a data-encodeable polymer for data encoding using repeatedly spaced chemically modifiable structures includes a polymer having a sequence of monomers in which one or more chemically modifiable structures are repeatedly repeated in a cluster of repeating units, each chemically modifiable structure of the one or more chemically modifiable structures being attached onto the nucleic acid polymer via a monomer, the one or more chemically modifiable structures being modifiable by a pulse of light or redox energy. In some embodiments, the cluster of repeating units comprises consecutively repeating units. In some embodiments, the cluster of consecutively repeating units comprises at least 10 repeating units. In some embodiments, the cluster of consecutively repeating units comprises at least 21 repeating units. In some embodiments, the cluster of consecutively repeating units comprises at least 625 repeating units. In some embodiments, the cluster of consecutively repeating units comprises at least about 0.6 kbp of repeating units. In some embodiments, the cluster of consecutively repeating units comprises at least about 1.2 kbp of repeating units.In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 1 micron or so over the length of the polymer. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 10 units. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 21 units. In some embodiments, the cluster of repeating units is repeatedly repeated at least every 625 units. In some embodiments, the cluster of repeating units includes at least about 0.6 kbp of repeating units. In some embodiments, the cluster of repeating units includes at least about 1.2 kbp of repeating units. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer. In some embodiments, the cluster of repeating units defines a data bit resolution defined by a distance of at least about 1 micron over the length of the polymer. In some embodiments, each chemically modifiable structure of the one or more chemically modifiable structures includes one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore. In some embodiments, a first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a first wavelength of light. In some embodiments, a second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a second wavelength of light.In some embodiments, a first chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at a second wavelength of light, and a second chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at a first wavelength of light. In some embodiments, the one or more chemically-modifiable structures include a photoconvertible fluorophore that exists in a first structural state having a first emission wavelength and is convertible to a second structural state having a second emission wavelength by a pulse of light or redox energy. In some embodiments, the conversion of the photoconvertible fluorophore from the first structural state to the second structural state is by a pulse of light at the first wavelength, and the photoconvertible fluorophore is convertible to a third structural state having a third emission wavelength by a pulse of light at the second wavelength. In some embodiments, one or more chemically modifiable structures of the polymer are repeatedly repeated every second monomer, every third monomer, every fourth monomer, every fifth monomer, every sixth monomer, every seventh monomer, every eighth monomer, every ninth monomer, every tenth monomer, every eleventh monomer, every twelfth monomer, every thirteenth monomer, every fourteenth monomer, every fifteenth monomer, every sixteenth monomer, every seventeenth monomer, every eighteenth monomer, every nineteenth monomer, every twentieth monomer, every twenty-first monomer, every twenty-second monomer, every twenty-third monomer, every twenty-fourth monomer, or every twenty-fifth monomer, as follows: In some embodiments, the polymer is an inorganic polymer.

[0010] The description and claims will be more fully understood with reference to the following drawings and data graphs, which are presented as exemplary embodiments and should not be construed as a complete description of the scope of the present disclosure. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 provides structural diagrams of examples of chemically modifiable structures that can be incorporated into nucleic acid polymers, according to various embodiments.

[0012] [Figure 2-1] 2A-2C present molecular structure diagrams of a set of exemplary chemically modifiable structures for use in writeable polymers, according to various embodiments. [Figure 2-2] 2A-2C present molecular structure diagrams of a set of exemplary chemically modifiable structures for use in writeable polymers, according to various embodiments. [Figure 2-3] 2A-2C present molecular structure diagrams of a set of exemplary chemically modifiable structures for use in writeable polymers, according to various embodiments.

[0013] [Figure 3-1] 3A-3C provide schematic diagrams of nucleic acids having chemically modifiable structures repeated repeatedly along the polymer, according to various embodiments. [Figure 3-2] 3A-3C provide schematic diagrams of nucleic acids having chemically modifiable structures repeated repeatedly along the polymer, according to various embodiments.

[0014] [Figure 4] FIG. 4 presents an exemplary reaction diagram for incorporating chemically modifiable structures into nucleic acid polymers, according to various embodiments.

[0015] [Diagram 5] FIG. 5 provides examples of functional groups that can be utilized to incorporate chemically modifiable structures into polymers, according to various embodiments.

[0016] [Figure 6] FIG. 6 presents a schematic of an exemplary method for encoding data by modifying chemically modifiable structures within clusters along a nucleic acid polymer, according to various embodiments.

[0017] [Figure 7] 7 shows examples of electrophoretic results of labeled DNA according to various embodiments, where lane 1 contains Cy5 labeled DNA and lane M contains Safegreen 1 kb DNA ladder.

[0018] [Figure 8] 8A-8C present results of DNA imaging of Cy5-labeled DNA before and after bleaching according to various embodiments. Figure 8A shows an image of Cy5-labeled DNA before bleaching, and Figure 8B shows an image of Cy5-labeled DNA after localized photobleaching. Figure 8C shows a plot showing two transverse profiles: before bleaching (1501) and after bleaching (1502).

[0019] [Figure 9-1] 9A-9D provide examples of data encoding by photobleaching of clusters of fluorophores on a DNA polymer, according to various embodiments. [Figure 9-2] 9A-9D provide examples of data encoding by photobleaching of clusters of fluorophores on a DNA polymer, according to various embodiments.

[0020] [Figure 10] 10A-10C provide examples of data encoded onto a DNA polymer containing 42 fluorophores per compartment, according to various embodiments.

[0021] [Figure 11] 11A-11C provide examples of data encoded onto a DNA polymer containing 21 fluorophores per compartment, according to various embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description Various embodiments are directed to polymeric data storage compositions and systems, methods of use thereof, and methods of synthesis thereof. In some embodiments, the writeable polymer is generated by generating a polymer with a repeatedly repeated chemically modifiable structure. In many embodiments, the chemically modifiable structure is incorporated into a biological polymer, such as DNA or RNA. In some embodiments, data is encoded into the polymer by modifying the chemically modifiable structure in a cluster. In many embodiments, the length of the cluster is dictated by the resolution provided by the writing device or method. The modification of the structure includes uncaging of the fluorophore, release of the quencher, uncaging of the DNA base, and / or photoconversion or photobleaching of the fluorescent dye, which can be performed chemically by utilizing light or redox energy.

[0023] When using single dye imaging to read and write data stored in biological polymers, single dye imaging, for example, when considering a single chromophore molecule attached to a single nucleobase of a DNA polymer, may require highly specialized and sensitive cameras due to the very weak fluorescence emitted by a single chromophore. Localizing the position of the dye in the image relative to other dyes that may be obscured by diffraction may require computer analysis. Furthermore, when using a single photomodifiable group as a bit, one bit must be separated from adjacent bits because the loss of one bit will cause an error in the series of bits. Furthermore, shining light on a single dye and modulating it may cause some stochastic fluctuations, which may cause errors including not modifying the chromophore when writing is intended. For example, the method may include removing the dye from a linker attached to the DNA backbone by light to encode digital information, and knowledge and manipulation of the position of the dye relative to the secondary structure of the DNA may be required in a nanopore sequencer. Thus, if the method involves nanopore sequencing, highly specialized and sensitive cameras and computer analysis may be required to localize the position of the dye in the image relative to other dyes that may be obscured by diffraction, as well as forward and reverse currents to move the DNA back and forth to process the information at the single nucleotide level, in addition to errors arising from stochastic fluctuations, all of which pose barriers to the adoption of such improved data storage technologies.

[0024] In response to such barriers in the art, methods are provided herein for the use of chemically modifiable structures (e.g., dye clusters in DNA or RNA) incorporated into biological polymers such as DNA or RNA to encode bits, where the clusters comprise a size larger than would be obscured by diffraction or other optical aberrations. Such embodiments may allow data encoded in the polymer to be read without the use of single molecule imaging methods, allowing the information encoded therein to be read using standard microscope cameras or detectors. In some embodiments, methods and compositions are provided herein that include clusters that may allow data to be written and read faster and at less cost. In some embodiments, such clusters are clusters of consecutively repeating units, or the clusters contain at least 10, 21, 625 units, or the clusters contain at least about 0.6 or 1.2 kbp of repeating units, or the clusters define a data bit resolution defined by a distance of at least about 0.3, 0.6, or 1 micron over the length of the polymer, allowing the data encoded in the polymer to be read by a standard microscope camera without the need to control the forward or reverse movement of the polymer in a nanopore sequencer and without computational analysis to account for diffraction. In some embodiments, the use of clusters of chemically modifiable or modified structures can eliminate such difficulties. In some embodiments, to overcome the problems caused by trying to read and write at the level of a single nucleobase, a stretch of polymer containing repeating (e.g., repeatedly spaced) modifiable residues can be written with variable resolution. In some embodiments, a single focus (e.g., area or spot) of light can contain multiple modifiable dyes. In some embodiments, stochastic variations in photomodulation can be averaged across multiple dyes, thereby reducing writing errors.

[0025] In some embodiments presented herein, the compositions and methods may include repeating dyes along a polymer (e.g., a DNA polymer strand). In such embodiments, the method including writing or reading data may include writing or reading data where each bit includes a cluster of residues (e.g., residues labeled with a dye). In some embodiments, precise positioning of the polymer relative to the light source may not be required. In some embodiments, data may be encoded along the length of the polymer, and precise positioning of the polymer relative to the light source may not be required, since encoding along the length of the polymer can start anywhere within the polymer and stop anywhere. In some embodiments, the method for reading or writing data may not include reversing direction, in which case nanopore sequencing is used. In some embodiments, the method that does not include reversing direction during nanopore sequencing may reduce the time for writing data. In some embodiments, not reversing direction in nanopore sequencing may simplify polymer design and required equipment.

[0026] In certain embodiments, fluorescently labeled non-covalent DNA assemblies can be assembled from synthetic oligonucleotides and assembled by hybridization. In such embodiments, the assembly assembled for use as a "molecular antenna" can contain fluorescent labels along the assembled segments, and digital data cannot be encoded because each assembled unit is the same. Such DNA assemblies for encoding digital information can contain more than one DNA polymer, and therefore digital data may not be stable due to the disassembly of DNA assemblies by dehybridization. For example, when using local modulation by dye fluorescence, information can be easily scrambled by dehybridization / rehybridization of the oligonucleotides contained in the assembly.

[0027] To enable the use of fluorescently labeled non-covalent DNA assemblies in the construction of data encoding polymers, various compositions of polymers containing photomodifiable groups are provided herein. In some embodiments, various methods are provided herein that include modifications to photomodifiable groups that include dyes. In some embodiments, polymers configured to encode digital information may contain clusters of photomodifiable groups.

[0028] In certain aspects, a method for encoding digital information onto a polymer may include synthesizing a polymer with different monomers that encode a binary bit per unit. Such aspects may include the use of light to remove the photocage group. In certain aspects, encoding occurs during assembly of the polymer into a specific sequence to encode the data.

[0029] In some embodiments presented herein, the method may include using clusters of the same monomers to encode digital information. In some embodiments presented herein, the method may include locally writing to an already assembled polymer. In some embodiments presented herein, the constructed polymer may not contain the encoded digital information, but rather may exist as an unwritten form of "tape". In some embodiments presented herein, the polymer may be assembled well before the writing of data, and the polymer may be used to encode any arbitrary bit sequence by optical writing along the polymer chain. In some embodiments, such a cluster is a cluster of consecutively repeating units, or the cluster comprises at least 10, 21, 625 units, or the cluster comprises at least about 0.6 or 1.2 kbp of repeating units, or the cluster defines a data bit resolution defined by a distance of at least about 0.3, 0.6, or 1 micron over the length of the polymer, thereby allowing the data encoded in the polymer to be read with a standard microscope camera without the need to control the forward or reverse movement of the polymer in a nanopore sequencer and without computer analysis to account for diffraction.

[0030] In certain embodiments, the compositions and methods may include incorporating photoswitchable fluorescent dyes into synthetic DNA oligonucleotides. In certain embodiments, oligonucleotides can be constructed by chemical synthesis and can be limited to a length of a few tens of nucleotides. In certain embodiments, the entire DNA can be illuminated with light (rather than locally) to change the color of the dye. In such embodiments, no digital information can be encoded. In such embodiments, fluorescent DNA bases may not be incorporated into longer DNA by polymerase enzymes.

[0031] In some embodiments presented herein, the compositions and methods may include long polymers that contain repeating copies of dyes. In some embodiments presented herein, the compositions and methods may include using clusters of dyes to encode digital information. In some embodiments presented herein, the compositions and methods may include repeating long polymers that contain many copies of photomodifiable groups. In some embodiments presented herein, the compositions and methods may include optical encoding of digital data by transforming clusters of such groups.

[0032] With reference now to the figures and data, compositions and systems for data storage utilizing nucleic acid polymers, methods for incorporating chemically modifiable groups into polymers, and methods for encoding are disclosed according to various embodiments. In some embodiments, the data storage system includes a polymer that includes a plurality of chemically modifiable structures along the nucleic acid polymer. Thus, the data-encodeable polymer is analogous to an encodable blank tape, and encoding into the data-encodeable polymer is achieved by selectively modifying the modifiable structures in clusters along the polymer chain, which can be achieved by any writing method for modification, including (but not limited to) localized light or redox energy. In many embodiments, a cluster is a localized domain defined by a data encoding resolution, and typically includes two or more modified structures that are modified in a writing step to encode a bit of data, with each modified structure extending from the polymer. In some embodiments, the modified structures of a cluster are uniformly modified, such that all of the chemically modifiable structures of the cluster undergo the same modification. In some embodiments, only a portion of the modified structures of the cluster are modified. In various embodiments, the chemically modifiable structure is a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore.Thus, in various embodiments, the chemically modifiable structure is modified by uncaging the nucleobase, uncaging the modified nucleobase, releasing the quencher, bleaching the fluorophore, or photoconverting the fluorophore.The modification of the chemically modifiable structure in a cluster can function as a data code, where the cluster of modified structures is similar to the data encoded in a "bit"; one modified structural state of the cluster is similar to "0" and the second modified structural state of the cluster is similar to "1".For example, in one embodiment, the chemically modifiable structure may include a plurality of caged fluorophores and a plurality of quenching fluorophores, which are provided in equal ratio; release of the cluster of only the quenchers of the cluster may be similar to a "0", and release of the cluster of the quenchers and uncaging of the cluster of the caged fluorophores may be similar to a "1". However, it should be understood that not only binary codes are possible, but data codes can be written in ternary, quaternary, or other numeric code, which can be done by utilizing multiple types of chemically modifiable structures or performing multiple writes / modifications. It should also be understood that data can also be coded by the spacing of the unconverted and converted clusters, since the unconverted clusters can be coded as "0" and the converted clusters can be coded as "1". Modification of the clusters of the chemically modifiable structures may be stable or permanent, allowing for long-term archiving, especially when kept in a dark storage location.

[0033] In many embodiments, the polymer incorporates residues with chemically modifiable structures incorporated or attached to the backbone, thus synthesizing a polymer with chemically modifiable structures that are repeatedly repeated along the polymer. In certain embodiments, the polymer is a nucleic acid polymer that incorporates nucleotides with chemically modifiable structures attached, and the chemically modifiable structures are repeatedly repeated along the polymer. In some embodiments, the polymer incorporates residues with reactive groups such that the residues with reactive groups are repeatedly spaced along the polymer. The reactive groups can be utilized to incorporate the chemically modifiable structures by a coupling reaction. In some embodiments, the chemically modifiable structures are provided in pairs (e.g., each reactive group incorporates a pair, and the pair consists of two chemically modifiable structures). Similarly, in some embodiments, each chemically modifiable structure (or pair of chemically modifiable structures) can include a reactive group that can be utilized to couple with one of the repeatedly spaced reactive groups of the polymer, thereby incorporating each set. In many embodiments, synthesis of a polymer with chemically modifiable structures or incorporation of chemically modifiable structures into a polymer converts the polymer into a data-encodeable polymer capable of encoding data into clusters. In such embodiments, the clusters of modified structures are defined by the resolution of the data writing process. In some such embodiments, between 2 and 2000 consecutive chemically modifiable structures are modified along the polymer into a cluster that resembles a bit of data. In some embodiments, between 2 and 20 modifiable structures are modified into a cluster. In some embodiments, between 2 and 20 modifiable structures are modified into a cluster.

[0034] In some embodiments, when data is encoded, consecutive chemically modifiable structures of a polymer are modified as a cluster, thus resulting in uniform modification within the cluster. In some embodiments, consecutive chemically modifiable structures of a polymer are modified simultaneously, resulting in uniform modification within each cluster. However, in some embodiments, when data is encoded as a cluster, only a portion of consecutive chemically modifiable structures are modified, resulting in a cluster having at least one modified structure within the cluster.

[0035] Many embodiments are directed to compositions of writeable polymers. Any suitable polymer may be utilized, including (but not limited to) biopolymers, organic polymers, and inorganic polymers. Biopolymers (and their analogs) include (but are not limited to) DNA, RNA, phosphorothioate DNA, glycerol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro-DNA, 2'-O-methyl RNA, locked nucleic acid (LNA), peptide chains, and peptoid chains. Nucleic acid polymers may be single-stranded or double-stranded. Additionally, nucleic acid polymers may utilize any enantiomer (e.g., d-DNA, l-DNA).

[0036] In some embodiments, the data-encodeable nucleic acid polymer comprises a plurality of chemically modifiable structures linked by a polymer backbone. In some embodiments, when data is encoded into clusters, a portion of the polymer between the clusters in which the data is encoded is left unmodified, resulting in spacing between the clusters. In some embodiments, a portion of the data polymer is unmodifiable and can serve as spacing between the clusters that remain unmodified. In various embodiments, the data-encodeable nucleic acid polymer may further comprise delimiters and / or data tags to label or identify the location of the data.

[0037] In some embodiments, data is encoded into a data-encodeable polymer using a data encoding procedure. Data encoding can be performed by selectively and locally modifying the chemically modifiable structures of the polymer with clusters, such that the encoded polymer contains clusters with a series of structural modifications that resemble a binary code of "0" and "1".

[0038] According to many embodiments, the data-encoded polymer is stored in the dark, in the absence of light to cause photobleaching. In some embodiments, the data-encoded polymer can be stored in an environment in which air or oxygen is excluded, thereby enhancing stability. Stabilizers such as (for example) alcohol, antioxidants, chelating agents, and biological inhibitors (e.g., nuclease inhibitors) can be included with the stored polymer. To read the data on the encoded nucleic acid polymer, nanopores capable of resolving the structural differences of the monomers can be utilized, such as, for example, Oxford Nanopore Technologies' PromethION, MinION, and GridION sequencing platforms (Oxford, UK). Nanopore devices can also be fabricated or manufactured for data reading. The nanopore can be composed of solid-state materials or can contain one or more proteins or other biologically produced molecules. Alternatively, when a fluorophore is utilized within the chemically modifiable structure, any suitable nanopore capable of detecting the fluorescence of the fluorophore can be utilized, such as Pacific Bioscience's Single Molecule, Real-Time (SMRT) sequencing platform (Menlo Park, Calif.), or a nanopore with plasmonic features to focus light energy near the pore.

[0039] In many embodiments, the chemically modifiable structures in the data-encodeable polymers are convertible into two different states that can be defined in a digital sense as 1 or 0. The encoded clusters are distinct from the unwritten state, and according to various embodiments, the differences are detectable by optical differences, current differences, or redox states, or magnetic states. In some embodiments, a series of encoded clusters along the encoded polymer chain results in a series of digital data. In some embodiments, the resolution of the method for encoding data defines the size of the clusters. The various methods of the present disclosure can be performed with much longer polymers than can be assembled with bottom-up approaches, thus resulting in denser data storage. Furthermore, data encoding can be performed more quickly than bottom-up approaches, since bits can be written at high speeds and no chemical assembly of molecules is required during the encoding process.

[0040] Generation of polymers with chemically modifiable structures Compounds and methods of synthesis according to embodiments of the present disclosure are based on generating polymers with residues having repeating chemically modifiable structures to generate data-encodeable polymers. Contiguous portions of multiple chemically modifiable structures along the polymer can be encoded as clusters and utilized as data "bits" such that the polymer can be encoded into a code (e.g., binary code). Each modified structure cluster (i.e., bit) can exist in two or more states: a first written state and at least a second written state. In some embodiments, the first written state is analogous to a "0" in binary code and the second written state is analogous to a "1". Data-encodeable polymers can be generated in long lengths (e.g., 5-50,000 residues having chemically modifiable structures, or longer) and can be made in bulk prior to data encoding.

[0041] In some embodiments, a data-encodable nucleic acid polymer comprises a sequence of nucleotides linked by a polymer backbone. In many embodiments, the sequence comprises nucleotides having repeatedly repeated chemically modifiable structures. For example, a sequence can be generated in which each thymine (T) has a chemically modifiable structure, and thymines can be regularly repeated. It should be understood that multiple chemically modifiable structures can be extended from any nucleotide structure, so long as the nucleotides are provided repeatedly. In various embodiments, a nucleotide structure with an added chemically modifiable structure is added to every other nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth ... th However, it should be understood that perfect regularity is not required and that the nucleotide structure to which the chemically modifiable structure is attached may be irregularly repeated.

[0042] In some embodiments, the chemically modifiable structures are designed to be modifiable by light pulses or by redox signals. In certain embodiments, the chemically modifiable structures are encoded as clusters according to spatial resolution. The spatial resolution depends at least in part on the data encoding mechanism. For example, if an optical light source and a device with a resolution of 20 nm are used to modify the chemically modifiable structures, the data encoded clusters can be approximately 59 bp, which is approximately 20 nm in length. This is smaller than the wavelength of light, but sub-wavelength resolution can be achieved by using methods such as plasmonics, zero-mode waveguides, or laser techniques such as STED. Furthermore, when a light source is applied to the polymer, all the chemically modifiable structures within the spatial resolution can be modified simultaneously, thus encoding a data bit cluster. The smaller the local energy resolution, the more modified structures are contained within the encoded cluster. Conversely, the greater the resolution, the fewer modified structures are contained within the encoded cluster. In some embodiments, the encoded clusters are separated by stretches of polymer that remain unmodified, and thus may contain the chemically modifiable structures in their original, unmodified state. In some embodiments, residues lacking chemically modifiable structures can be utilized as spacers between clusters. In various embodiments, the data-encodeable polymer can further include delimiters and / or data tags for labeling data, each of which can be provided by a specific residue sequence.

[0043] In many embodiments, data is encoded into clusters of modified structures along a nucleic acid polymer modified by light or redox. The starting and encoded states can be distinguished by changes in their properties within the polymer. For example, the chemically modifiable structures can have changes in fluorescence intensity or wavelength, changes in structure that change their size / shape so that they can be distinguished by the nanopore current, changes in magnetic or spin properties, or changes in redox properties. To encode data in binary form according to some embodiments, the chemically modifiable structures can be changed from a modifiable structure to a first modified structure (similar to 0) and / or to a second modified structure (similar to 1). This can be achieved by including two chemically modifiable structures attached to unique residue structures along the nucleic acid polymer (e.g., one structure extending from a T and the second structure extending from a C), or two chemically modifiable structures linked and attached at one attachment point (e.g., both structures extending from a T). An example of this case can include photobleaching of two differently colored fluorescent dyes attached to the same nucleotide. In some embodiments, the chemically modifiable structure is a single molecule that can be converted from a starting state to two different resulting structures, for example by photoconversion of a cyanine dye. In some embodiments, another molecular approach is to convert the two different chemically modifiable structures into new chemical groups that can each be detected independently by fluorescent labels or redox groups. In some embodiments, the assembled data-encodeable polymer contains such convertible structures throughout the entire chain to ensure that a bit is always encoded regardless of the position of the polymer relative to the writing device.

[0044] FIG. 1 provides examples of chemically modifiable structures that can be attached to residues and incorporated into polymers. The chemically modifiable structures allow the polymer to be data-encodeable by localized light or redox signals. According to various embodiments, the chemically modifiable structures include one or more chemical groups that can be structurally altered from a first structural state (e.g., caged) to a second structural state (e.g., uncaged) by controlled reaction chemistry (e.g., light energy or redox energy). In some embodiments, the chemically modifiable structures incorporate a fluorophore that is caged, where the caged fluorophore is in a first structural state and release of the cage results in a second structural state. In some embodiments, the chemically modifiable structures incorporate a quenching fluorophore, where the quenching fluorophore has a first structural state and release of the quencher results in a second structural state, typically resulting in brighter fluorescence. In some embodiments, the chemically-modifiable structure incorporates a fluorophore that can be converted from a first state having a particular fluorescent quality (e.g., a first emission or absorption wavelength) to a second state having a second distinguishable fluorescent quality (e.g., a second emission or absorption wavelength). In some embodiments, the chemically-modifiable structure incorporates two structurally alterable groups, resulting in a set of structures that can be modified to at least two distinct states.

[0045] Each of Figures 2A-2C provides an example of a set of chemically modifiable structures that can be utilized to encode data, according to various embodiments. In many embodiments, the chemically modifiable structures can be incorporated along the polymer chain. As shown in Figures 2A-2C, these chemically modifiable structures have dual-bit write capabilities. Thus, each of the set of chemically modifiable structures is added to a residue in an unwritten state and then transformed to a first written state and / or a second written state. "X" indicates a bond to the polymer residue.

[0046] The example in FIG. 2A is a set of chemically modifiable structures that includes two caged fluorophores. In some embodiments, the two-fluorophore adduct includes two fluorophores that are within approximately 30 angstroms of each other. In this example, two fluorophores of different colors are utilized in the set of chemically modifiable structures: coumarin and Tokyo green. Additionally, each fluorophore is caged with a caging group. Before data writing, there is little or no fluorescence emission by the polymer due to the caging of the fluorophores. To encode a bit of data, one or more pulses of violet light (e.g., approximately 430 nm) are used to uncage only the blue coumarin fluorophore, resulting in blue fluorescence. Alternatively, one or more pulses of UV light (e.g., approximately 365 nm) are used to uncage both the blue coumarin fluorophore and the Tokyo green fluorophore, resulting in a blue emission signal from the coumarin plus a green emission signal from the Tokyo green. Alternatively, a double pulse of violet (e.g., approximately 430 nm) and UV (e.g., approximately 365 nm) light can uncage both the blue coumarin fluorophore and the Tokyo green fluorophore, resulting in a blue emission signal from the coumarin plus a green emission signal from the Tokyo green. When both fluorophores are uncaged, energy can be donated from the blue fluorophore to the green fluorophore, resulting in a Förster energy transfer, enhancing the green fluorescence and reducing the blue fluorescence. Thus, in this approach, data can be encoded as a binary code of clusters of blue and green emission signals along the polymer. A set of chemically modifiable structures can be incorporated into a nucleotide or synthesized as part of a nucleotide and then incorporated into a polymer by biological or chemical synthesis. Alternatively, a set of chemically modifiable structures can be incorporated into a polymer via reactive residues attached to the backbone as indicated by X in the drawing.Although a few examples are provided, it will be understood that any suitable fluorophore and cage can be used in accordance with various embodiments.

[0047] A variety of photoremovable groups can be incorporated into the caged fluorophores (see, e.g., Y. Zhao, et al., J Am Chem Soc. 2004; 126:4653-63, the disclosure of which is incorporated herein by reference). As can be seen in the figure, each fluorophore has a caging component linked by an energy (e.g., light or redox) cleavable linker (e.g., an ether or carbonate or carbamate group). The fluorophore can be directly attached to the polymer backbone via a reactive group on a residue attached to the backbone. Although a few examples are provided, it will be understood that any suitable photoremovable group and fluorophore can be used in accordance with various embodiments.

[0048] The example of FIG. 2B is a set of chemically modifiable structures that include photoconvertible fluorophores that can be converted by light of a particular wavelength to emit a second and / or third fluorescent wavelength. In some embodiments, clusters of photoconvertible fluorophores can be utilized in a manner similar to a "bit" of data, thereby allowing conversion from a first structural state (e.g., a first emission wavelength) to a second structural state (e.g., a second emission wavelength) similar to a "0" or "1" digital bit designation. In some embodiments, photoconvertible fluorophores can be converted from a first emission state (e.g., a first emission wavelength) to a second emission state (e.g., a second emission wavelength) and further to a third emission state (e.g., a third emission wavelength), thereby allowing a ternary code or a positively written binary code (i.e., the first emission state is an unwritten state, the second emission state is similar to a "0", and the third emission state is similar to a "1"). The fluorescent state of the bits can be detected using a nanopore device capable of detecting the fluorescence of individual fluorophore molecules.

[0049] A variety of photoconvertible fluorophore groups are available (see, for example, TJ Chozinski, LA Gagnon, and JC Vaughan, FEBS Lett. 2014; 588:3603-12, the disclosure of which is incorporated herein by reference). As can be seen in the example of FIG. 2B, a heptamethine cyanine dye can be converted to either a pentamethine cyanine dye or a trimethine cyanine dye by a pulse of light of a specific wavelength. In this example, a heptamethine cyanine dye with a 7-carbon alkene-containing chain emits near-infrared fluorescence. Light energy at approximately 740 nm modifies the alkene-containing chain of the heptamethine cyanine dye, resulting in a pentamethine cyanine dye with a 5-carbon chain. The pentamethine cyanine dye emits red fluorescence. Furthermore, light energy at approximately 638 nm modifies the alkene-containing chain of the heptamethine cyanine dye or the pentamethine cyanine dye, resulting in a trimethine cyanine dye with a three-carbon chain. The trimethine cyanine dye emits yellow / green light. Thus, in this approach, data can be encoded as a binary code of red and yellow / green light-emitting signals. A set of chemically modifiable structures can be incorporated into a nucleotide or synthesized as part of a nucleotide, and then incorporated into a polymer by biological or chemical synthesis. Alternatively, a set of chemically modifiable structures can be incorporated into a polymer via a reactive residue attached to the backbone, which is indicated by X in the diagram. Although a few examples are provided, it is understood that any suitable photoconvertible fluorophore can be used according to various embodiments.

[0050] The example in FIG. 2C is a set of chemically modifiable structures that includes a combination of a releasable quencher and a caged fluorophore. In some embodiments, the two-fluorophore bit includes two fluorophores that are within approximately 30 angstroms of each other. In this example, two fluorophores of different colors are utilized in the set of chemically modifiable structures: unmodified coumarin and photocaged Tokyo green. Additionally, a photoreleasable fluorescence quencher can be incorporated near the two dyes with a photocleavable linker (and in this example is attached to the coumarin blue fluorophore). Before data writing, there is little or no fluorescence emission by the polymer due to the caging of the fluorescence quencher and fluorophore. To write a bit of data, one or more pulses of violet light (e.g., approximately 430 nm) are used to release only the quencher, resulting in blue fluorescence from the blue coumarin. Alternatively, one or more pulses of UV light (e.g., about 365 nm) are used to release the quencher and uncage the Tokyo green fluorophore, thereby generating a blue emission signal from coumarin plus a green emission signal from Tokyo green. Alternatively, a dual pulse of violet light (e.g., about 430 nm) and UV light (e.g., about 365 nm) releases the quencher and uncages the Tokyo green fluorophore, thereby generating a blue emission signal from coumarin plus a green emission signal from Tokyo green. A set of chemically modifiable structures can be incorporated into a nucleotide or synthesized as part of a nucleotide, and then incorporated into a polymer by biological or chemical synthesis. Alternatively, a set of chemically modifiable structures can be incorporated into a polymer via a reactive residue attached to the backbone, as indicated by X in the drawings. Although a few examples are provided, it is understood that any suitable fluorophore and cage can be used according to various embodiments.

[0051] A variety of quencher groups can be utilized (see, for example, JR Lakowicz, (Ed.). (2013). Principles of fluorescence spectroscopy. Springer science & business media. pp. 277-330 "Quenching of Fluorescence"; and MK Johansson, Methods Mol Biol. 2006; 335:17-29, the disclosures of which are each incorporated herein by reference). As can be seen from the figures, the quencher has a linker (e.g., a nitrobenzyl group) that is cleavable using light energy. The quencher can be attached directly to the polymer backbone, to a residue attached to the backbone, or to a fluorophore. Although a few examples are provided, it is understood that any suitable quencher, release mechanism, and fluorophore can be used in accordance with various embodiments.

[0052] Some embodiments are directed to encodable polymers. In many embodiments, the encodable polymer comprises a plurality of chemically modifiable structures that are repeatedly repeated. In some embodiments, the plurality of chemically modifiable structures along the polymer chain are modified as clusters, such that each cluster is one bit of encodable data. In many embodiments, the clusters are defined by the resolution of the data encoding device. For example, if the resolution of the data encoding device is 20 nm, each cluster is defined to be 20 nm long, approximately 59 bp. In various embodiments, the clusters are defined to be any length between 1 nm and 400 nm. In certain embodiments, the clusters are defined to be 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, or 400 nm long. Furthermore, a cluster can incorporate any number of modified structures depending on the repeat interval of the structure and the data encoding spatial resolution. In certain embodiments, the repeat interval of the chemically modifiable structure is every second residue, every third residue, every fourth residue, every fifth residue, every sixth residue, every seventh residue, every eighth residue, every ninth residue, every tenth residue, every eleventh residue, every twelfth residue, every thirteenth residue, every fourteenth residue, every fifteenth residue, every sixteenth residue, every seventeenth residue, every eighteenth residue, every nineteenth residue, every twentieth residue, every twenty-first residue, every twenty-second residue, every twenty-third residue, every twenty-fourth residue, every twenty-fifth residue, or any other suitable interval to suit the data encoding spatial resolution. In certain embodiments, the chemically modifiable structure is irregularly repeated.

[0053] In various embodiments, the polymer incorporating the chemically modifiable structure can be of any length, for example, from as little as 15 residues to more than 100,000 residues. In certain embodiments, the polymer is longer than 100 residues, longer than 200 residues, longer than 300 residues, longer than 400 residues, longer than 500 residues, longer than 1000 residues, longer than 5000 residues, longer than 10,000 residues, longer than 50,000 residues, or longer than 100,000 residues. The maximum length is only limited by the stability of the polymer, the method used to synthesize the polymer, and the method used to read the encoded data. Longer chains containing more bits have the advantage that more data is contained per molecule.

[0054] An exemplary schematic of a nucleic acid containing chemically modifiable structures along the polymer is provided in FIG. 3A. In this example, the polymer is linked to thymine on one strand of a double-stranded polymer, with every 3 nucleotide rd The nucleic acid sequence includes an adduct that includes two chemically modifiable structures (e.g., a green dye and a red dye, as shown) that are repeated on a nucleotide sequence. Although the adducts are shown extending from thymine, these adducts may extend from any nucleotide, including (but are not limited to) thymine (T), guanine (G), cytosine (C), adenine (A), uracil (U), or any other residue that can be polymerized into a nucleic acid polymer. Additionally, although the adducts are shown extending from every third nucleotide, these adducts may be repeated and spaced in any manner such that at least a plurality of consecutive adducts are modified as a cluster by the data encoding mechanism.

[0055] An exemplary schematic of a nucleic acid containing chemically modifiable structures along the polymer is provided in Figure 3B. In this example, the polymer contains adducts containing a single chemically modifiable structure (e.g., a green or red dye) that are each linked to a thymine and are repeated every third nucleotide, where the top strand adduct incorporates a chemically modifiable structure that is a green dye and the bottom strand adduct incorporates a chemically modifiable structure that is a red dye.

[0056] An exemplary schematic of a nucleic acid containing chemically modifiable structures along the polymer is provided in Figure 3C. In this example, the polymer contains adducts each linked to a thymine and containing a single chemically modifiable structure repeated every third nucleotide, where the top strand adduct incorporates a chemically modifiable structure called a photocaged fluorescein dye and the bottom strand adduct incorporates a photocaged rhodamine dye as the chemically modifiable structure.

[0057] Many embodiments are also directed to nucleic acid polymers that further incorporate one or more of spacers, delimiters, and data tags. According to various embodiments, spacers are residues incorporated into the polymer that provide spacing between chemically modifiable structural bits. In some embodiments, data-encoding nucleic acid polymers repeatedly utilize the same residue structure for each and every spacer. However, in some embodiments, the nucleic acid polymer utilizes two or more different residues as spacers. Any suitable residue can be utilized as a spacer, including naturally occurring nucleobases, non-natural nucleobases, tetrahydrofuran abasic residues, and / or ethylene glycol residues.

[0058] According to various embodiments, a delimiter is a residue that indicates a boundary. In some embodiments, a delimiter is used to separate two adjacent data fields. Any suitable residue that lacks the ability to incorporate bits can be used as a delimiter, including naturally occurring nucleobases, non-natural nucleobases, tetrahydrofuran abasic residues, and / or ethylene glycol residues.

[0059] In some embodiments, a data tag is a continuous string of monomers (typically four or more residues) that indicates a particular piece of data. For example, a data tag can indicate the type of data, the date, the data source, or any other information. Any suitable residue that lacks the ability to incorporate a bit can be utilized as a data tag residue, including naturally occurring nucleobases, non-natural nucleobases, tetrahydrofuran abasic residues, and / or ethylene glycol residues.

[0060] In some embodiments, an existing nucleic acid molecule can be utilized as a substrate for adding a chemically modifiable structure. In some embodiments, a nucleotide having a chemically modifiable structure is incorporated into the polymer. In some embodiments, a chemically modifiable structure is incorporated into a nucleic acid base and / or an existing nucleic acid molecule by chemical reaction with the nucleic acid base (see, e.g., FIG. 4). In some embodiments, a polymerase extension is performed to incorporate a residue having a reactive functional group into the nucleic acid polymer (see, e.g., FIG. 5). In some embodiments, a polymerase extension is performed to incorporate a residue having a chemically modifiable structure already attached or synthesized within the residue. Any suitable means for performing polymer extension can be utilized. In some embodiments, when performing polymer extension, one or more nucleoside triphosphates are modified with a functional group or a chemically modifiable structure, thus incorporating a functional group or a chemically modifiable structure into the nucleic acid polymer throughout the polymer chain. The incorporation of a reactive functional group allows the incorporation of a set of chemically modifiable structures for each of the functional groups along the polymer. Methods for encoding and decoding data

[0061] Some embodiments are directed to encoding and decoding data on polymers using chemically modifiable structures. In many embodiments, data-encoding polymers are prepared such that the chemically modifiable structures are repeated repeatedly along the polymer. The prepared writable polymers may also have spacers, delimiters, and data tags as described herein. To write data onto the polymer, according to various embodiments, individual strands are passed through a device having a nanopore. Various nanopore devices allow the chemically modifiable structures to undergo conformational changes as they pass through the pore (see, for example, D. Garoli, et al., Nano Lett. 2019; 19:7553-7562; and M. Rahman, et al., Lab Chip. 2021; 21:3030-3052, the disclosures of which are each incorporated herein by reference). When a photocage or cleavable linker travels along the strand and is photoexcited, cleavage of the chemical bond occurs, thereby resulting in structural modification. Such devices may include structures in the vicinity of the nanopore that amplify the optical or redox energy to a highly localized location, including (but not limited to) plasmonic amplifiers such as metallic bowties, metallic nanorods, or zero-mode waveguides (see, e.g., JD Spitzberg, et al., Adv Mater. 2019; 31: e1900422, the disclosure of which is incorporated herein by reference).

[0062] By pulsing energy into such plasmonic structures, resolutions ranging from as small as one nanometer to as large as the wavelength of light can be obtained. Alternatively, highly localized illumination can be achieved using the use of two lasers by the STED technique (see, for example, SJ Sahl and SW Hell High-Resolution 3D Light Microscopy with STED and RESOLFT. 2019 Aug 14. In: Bille JF, editor. High Resolution Imaging in Microscopy and Ophthalmology: New Frontiers in Biomedical Optics. Cham (CH): Springer; 2019. Chapter 1, the disclosure of which is incorporated herein by reference). Any resolution achievable by the device and desired for the size of the clusters can be utilized. In some embodiments, the size of the clusters is dictated by the resolution of the data encoding device. In many embodiments, the resolution of the data encoding device is determined by the preferred size of the clusters.

[0063] In some embodiments, the act of encoding is accomplished by passing a polymer through a plasmonic nanopore and using pulses of light at two wavelengths to convert two different groups to 1 or 0 states. In many embodiments, the encodable polymer is stretched and two different wavelengths of light are applied locally to the chemically modifiable structures using subwavelength focusing techniques such as STED. In various embodiments, a continuous string of chemically modifiable structures comes within the resolution of the light energy, and then they are uniformly converted as a group because they all receive the same pulse of light, resulting in the encoding of a cluster bit. Other methods of moving DNA through a writing mechanism are also contemplated, such as flowing stretched DNA through a capillary.

[0064] It is understood that the modified structures within the modified cluster can be completely and uniformly modified. For example, if all 10 green dyes and all 10 red dyes are within the resolution of a single energy pulse with a resolution of 20 nm, and the green dye bleaching encodes "1", a pulse of light can convert all 10 green dyes within that spatial resolution to a dark state, thereby indicating a pure red fluorescent signal at that spot. However, in some embodiments, a pulse that converts only 50% of those green dyes to a dark state still produces a detectable fluorescence difference (red to green ratio 2:1), thus providing encoded data. Thus, according to various embodiments, any detectable change within the encoded cluster can be utilized as encoded data. In some embodiments, the data encoding results in at least one modification of the chemically modifiable structure within the encoding resolution. In some embodiments, the data encoding results in at least a plurality of modifications of the chemically modifiable structure within the encoding resolution.

[0065] In some embodiments, modification of the chemically modifiable structure does not need to be performed for every repeat unit of the polymer. Units can be skipped during the encoding process, resulting in spaces between encoded clusters. When decoding a polymer with skipped units, such skipped units can be considered as blanks or nulls, and the reader advances to the next fluorescent position to find the next cluster bit in the sequence. The skipping of repeat units during encoding and decoding can be performed intentionally to align the bit encoding to best suit the resolution of the encoding method; skipping can also occur randomly due to stochastic movement and changes in the speed at which molecules pass through the pore.

[0066] In many embodiments, the encoding device is provided with software code for encoding data into the polymer. The encoding device is then directed by this code to provide pulses of energy controlled by time and / or wavelength to selectively modify the chemically modifiable structures of the polymer to obtain a data code (e.g., a binary code). Once the data code has been encoded into the polymer, the polymer can be stored in the dark and by any suitable means to maintain the integrity of the polymer molecules. For example, the data-encoded polymer can be stored dried, as a precipitate, or in a suitable solution, at room temperature or at low temperature (e.g., -20°C). Stabilizers such as (for example) alcohol, antioxidants, chelating agents, and biological inhibitors (e.g., nuclease inhibitors or protease inhibitors) can be included with the stored polymer.

[0067] Polymers are the ones that store data most efficiently at the single molecule level, thereby providing the highest potential density of information. However, in some embodiments, if data redundancy is required for better accuracy of data storage, multiple polymers can be used to redundantly encode the same data in each of the multiple polymers. Error correction algorithms for digital data storage are already well developed, and some of these algorithms can be applied to the present approach (see J. Li, et al., IEEE Transactions on Emerging Topics in Computing. 2021; 9:651-663, the disclosure of which is incorporated herein by reference).

[0068] Highly localized optical excitation can be achieved by subwavelength focusing strategies with specialized microscopes such as STEDX, or by using nanoplasmonic structures such as bowties, or by using zero-mode waveguides (see Y. Fang and M Sun, Light Sci Appl. 2015; 4: e294; and X. Shi, et al. Small. 2018; 14: e1703307, the disclosures of which are each incorporated herein by reference). The timing of the light pulses and the controlled passage of the writeable polymer can be coordinated with appropriate intervals so that data is encoded with fidelity.

[0069] In FIG. 6, a schematic diagram of a method for encoding a data-encodable nucleic acid molecule by an encoding device with a resolution that results in a cluster of modified structures is presented. In this example, a nucleic acid polymer is constructed to contain side chains at thymidine residues, where the side chains have fluorescein (green) and rhodamine (red) dyes linked to the amine groups of the thymine base. To encode data, the nucleic acid molecule can be passed through a plasmonic nanopore with an optical resolution of 5 nm. As the DNA passes through the pore, the fluorescein (green) and rhodamine (red) dyes in the cluster can be modified to the written state, thereby encoding a "0" or a "1". As shown in FIG. 6, a pulse of light with a wavelength that excites the fluorescein is applied to the plasmonic structure, resulting in a high-energy focus on a cluster of five chemically modifiable structures. The light energy bleaches the fluorescein in the cluster, resulting in an encoded cluster of rhodamine fluorescence with diminished fluorescein fluorescence. Additionally, as the nucleic acid passes through the nanopore, a pulse of light at a wavelength that excites rhodamine is directed at the plasmonic structure, resulting in high energy focusing on a cluster of five chemically modifiable structures. The light energy bleaches the rhodamine in the cluster, resulting in an encoded cluster of fluorescein fluorescence and diminished rhodamine fluorescence. By strategically photobleaching the fluorescein or rhodamine dyes, a binary code can be written, represented by green (fluorescein) and red (rhodamine) clusters. Unwritten regions (shown as green and red fluorescence in Figure 6) can be left between the encoded clusters, resulting in spacers between clusters that are data encoded.

[0070] In some embodiments, the encoded polymer contains clusters of structures that have been converted to "1" or "0" states, with non-encoded (unconverted) structures interspersed between the encoded clusters. In many embodiments, the clusters are read by observing the train of "1" and "0" state clusters along the polymer. Observation can be done by passing the polymer through a plasmonic nanopore and observing the change in fluorescence of the encoded bits as the polymer chain passes. Alternatively, the polymer can be passed through a zero-mode waveguide in a nanowell and subsequently imaged, similar to its use in SMRT sequencing. Another approach is to stretch or comb the polymer chain and image the fluorescence change along the linear stretched chain.

[0071] In many embodiments, any suitable nanopore capable of reading the fluorescence of a single fluorophore or analyzing structural differences can be utilized to decode data on an encoded nucleic acid polymer. In certain embodiments, the device is capable of both encoding and decoding a nucleic acid polymer. In certain embodiments, a single nanopore has dual functionality for both encoding and decoding from the polymer, although some devices may include separate nanopores to perform encoding and decoding. A variety of nanopore devices are commercially available, or alternatively, nanopore devices for encoding and / or decoding data can be fabricated or manufactured. The nanopore may be composed of a solid state material.

[0072] As an alternative to using a nanopore device for decoding, a polymer can be stretched and imaged using a device capable of decoding or reading fluorophores along the stretched polymer. Methods for stretching or combing polymers (especially nucleic acids) are known to those skilled in the art (see, for example, ZE Nazari and L. Gurevich, J. Self-Assembly and Molecular Electronics 2013; 1:125-148; A. Kaykov, et al., Sci Rep. 2016; 6:19636, the disclosures of which are incorporated herein by reference). Alternatively, in some embodiments, super-resolution microscopy can be used (by STED techniques or other known methods) to achieve high spatial resolution. Imaging a single polymer chain by STED can provide a series of dye colorations that represent a series of bits. The method can be automated for high throughput by stretching many chains in one field of view and using automated imaging software to read the series of dyes (bits) and convert them into digital information.

[0073] Energy pulses are used to convert or otherwise modify dyes and / or quenchers within the polymer, resulting in detectable structural changes in the repeating units of the polymer. Thus, in addition to the fluorescence methods described above, data-encoded polymers can also be read by non-fluorescence methods. An example is nanopore sequencing by using alterations in ion flow. Another method involves reading the optical absorption or other spectral signatures (e.g., vibrational modes, etc.) of the modified dyes as the strand passes through the pore. Another method involves reading the redox signal as the strand passes through the pore.

[0074] In certain aspects, provided herein are various methods of encoding data onto various writeable polymers, including providing a data-encodeable nucleic acid polymer having a sequence of nucleotides in which one or more chemically-modifiable structures are repeatedly repeated, where each of the one or more chemically-modifiable structures is attached onto the nucleic acid polymer, where the one or more chemically-modifiable structures are capable of being modified to a second structural state by a pulse of light or redox energy; and utilizing a data encoding device to selectively modify a subset of the one or more chemically-modifiable structures along the nucleic acid polymer to the second structural state, resulting in a polymer encoded with data defined by the modified structures in a plurality of clusters, where a length of each cluster of the plurality of clusters is determined by a spatial resolution.

[0075] In some embodiments, the spatial resolution is defined by the resolution of the laser (e.g., laser spot size or laser spot). In some embodiments, the spatial resolution includes the minimum length of a polymer that contains one bit (e.g., a single bit). In some embodiments, the spatial resolution includes the length of a polymer that can be written as a single bit. In some embodiments, the spatial resolution includes the minimum length of a polymer that can be written as a single bit. In some embodiments, the spatial resolution includes the length of a polymer that can be written as a single bit.

[0076] In some embodiments, each chemically-modifiable structure of the one or more chemically-modifiable structures comprises a photobleachable fluorophore.

[0077] In some embodiments, the selectively modifying step comprises photobleaching clusters of fluorophores. In some embodiments, the selectively modifying step comprises locally photomodifying a single fluorophore-labeled DNA polymer.

[0078] In some embodiments, the data-encodable nucleic acid polymer may comprise a double stranded data-encodable nucleic acid polymer. In some embodiments, the data-encodable nucleic acid polymer may comprise a double stranded DNA polymer. In some embodiments, the data-encodable nucleic acid polymer may comprise a size of about 1 kbp to about 100 kbp. In some embodiments, the data-encodable nucleic acid polymer may comprise a size of about 40 kbp.

[0079] In some embodiments, the data-encodable nucleic acid polymer may include multiple fluorophores. In some embodiments, the data-encodable nucleic acid polymer may include multiple fluorophores, where uridines may be labeled with fluorophores. In some embodiments, the fluorophores may be repeatedly spaced along the polymer. In some embodiments, a fluorophore may be present every 1, 2, 3, 4, 5, or more bp. In some embodiments, a fluorophore is present every 4 bp.

[0080] In some embodiments, a data-encodable nucleic acid polymer can be labeled with about 625 StarRed fluorophores per μm (on uridines every 4 bp). In some embodiments, a 40 kbp double stranded DNA polymer can be labeled with about 625 StarRed fluorophores per μm (on uridines every 4 bp) and stretched to a length of approximately 16 μm on glass, as shown in Figure 9A. The x in Figure 9A represents either an adenine, cytosine or guanine residue.

[0081] In some embodiments, the selectively modifying step may include photobleaching using a laser. In some embodiments, the selectively modifying step may include photobleaching using a laser, where photobleaching occurs at specific locations along the DNA polymer to encode data. In some embodiments, a laser can be focused onto a spot (e.g., a laser spot) on the DNA polymer that includes a fluorophore. In some embodiments, a laser can be focused onto a spot (e.g., a laser spot) on the DNA polymer that includes a fluorophore, where a laser is used to bleach (e.g., photobleach) the fluorophore at specific locations along the polymer.

[0082] In some embodiments, as shown in Figure 9A, a 40 kb region of double-stranded DNA can be constructed to contain approximately 10,000 fluorescent dyes (StarRed, conjugated to deoxyuridine) that are repeated every 4 base pairs in the sequence.

[0083] In some embodiments, the dye may include a fluorescent dye. In some embodiments, the fluorescent dye may include an excitation maximum at about 300 nm to about 800 nm. In some embodiments, the fluorescent dye may include an excitation maximum at about 638 nm. In some embodiments, the fluorescent dye may include an emission maximum at about 300 nm to about 800 nm. In some embodiments, the fluorescent dye may include an emission maximum at about 655 nm. In some embodiments, the dye may include an excitation maximum at about 638 nm and an emission maximum at about 655 nm. In some embodiments, the dye may include an excitation maximum at about 638 nm and an emission maximum at about 655 nm. In some embodiments, the dye may include an excitation maximum at about 638 nm and an emission maximum at about 655 nm. In some embodiments, the dye may include an excitation maximum at about 638 nm and an emission maximum at about 655 nm.

[0084] In some embodiments, DNA can be stretched on glass. In some embodiments, DNA can be stretched on glass, where the stretched DNA comprises a length. In some embodiments, the length of the stretched DNA can be about 0.1 μm to about 1000 μm. In some embodiments, the length of the stretched DNA can be about 1 μm to about 100 μm. In some embodiments, the length of the stretched DNA can be greater than about 1000 μm. In some embodiments, the length of the stretched DNA can be about 16 μm.

[0085] Various methods are described herein that include stretching DNA. In some embodiments, the method may include stretching DNA and fixing the DNA on a surface. In some embodiments, the method may include stretching DNA and fixing the surface, including glass. In some embodiments, the method may include observing a single DNA molecule. In some embodiments, the method for stretching and fixing DNA may include Genomic Vision's FiberComb® Molecular Combing System.

[0086] In some embodiments, the stretched, writeable DNA polymer may comprise a density of about 10 fluorophores per μm to about 1000 fluorophores per μm. In some embodiments, the stretched, writeable DNA polymer may comprise a density of about 500 fluorophores per μm to about 600 fluorophores per μm. In some embodiments, the stretched, writeable DNA polymer may comprise a density of more than about 1000 fluorophores per μm. In some embodiments, the stretched, writeable DNA polymer may comprise a density of less than about 1000 fluorophores per μm. In some embodiments, the stretched, writeable DNA polymer may comprise a density of approximately 625 fluorophores per μm.

[0087] In some embodiments, the selectively modifying step comprises the use of a laser. In some embodiments. In some embodiments, the selectively modifying step comprises the use of a laser comprising a wavelength of about 640 nm or longer. In some embodiments, the selectively modifying step comprises the use of a laser comprising a wavelength of 640 nm or shorter. In some embodiments, the selectively modifying step comprises the use of a laser comprising a wavelength of about 640 nm.

[0088] In some embodiments, the selectively modifying step may include using a laser to form a photobleached spot on the stretched writeable polymer. In some embodiments, the stretched writeable polymer may be a DNA polymer. In some embodiments, the spot may include a diameter of about 1 μm. In some embodiments, the spot may include a diameter of more than about 1 μm. In some embodiments, the spot may include a diameter of less than about 1 μm.

[0089] In some embodiments, the selectively modifying step may comprise a duration of less than about 25 milliseconds. In some embodiments, the selectively modifying step may comprise a duration of greater than about 25 milliseconds. In some embodiments, the selectively modifying step may comprise a duration of about 25 milliseconds.

[0090] In some embodiments, the selectively modifying step may include using a laser comprising a laser power. In some embodiments, the laser power may be less than about 60mW. In some embodiments, the laser power may be greater than about 60mW. In some embodiments, the laser power may be about 60mW.

[0091] In some embodiments, the selectively modifying step may include automatically stepping a laser light from one location (eg, a photobleached spot) to the next location.

[0092] Images acquired before and after the step of selectively modifying by photobleaching with a laser according to some embodiments are shown in Figure 9B as "before" and "after", respectively. An average trace of ten photobleached DNAs is shown in Figure 9C. A second example of photobleaching DNA to encode the character "IO" (1001001 1001110) is shown in Figure 9D.

[0093] In certain aspects, various methods are provided herein for encoding data on polymers using prefabricated fluorophore clusters and for reading data.In some embodiments, the polymer is a DNA polymer.In some embodiments, the polymer can be stretched and fixed on a substrate.In some embodiments, the polymer can be stretched and fixed on a substrate, and the polymer can include alternating sections.

[0094] In some embodiments, the stretched DNA polymer may comprise a size of about 6 kb, as shown in Figure 10A. In some embodiments, the size of the stretched DNA polymer may be greater than about 6 kb. In some embodiments, the size of the stretched DNA polymer may be less than about 6 kb.

[0095] In some embodiments, the alternating sections may comprise DNA residues. In some embodiments, the alternating sections may comprise fluorophores. In some embodiments, the alternating sections may comprise DNA residues and fluorophores. In some embodiments, the fluorophores may comprise Cy5 fluorophores. In some embodiments, the DNA residues may comprise Cy5-labeled uridine.

[0096] In some embodiments, the alternating segments may comprise a size of about 0.1 kb or less. In some embodiments, the alternating segments may comprise a size of about 2 kb or more. In some embodiments, the alternating segments may comprise a size of 1.2 kb, as depicted in FIG. 10A. In some embodiments, the alternating segments may comprise a size of 0.6 kb, as depicted in FIG. 11A.

[0097] In some embodiments, the length of the alternating sections may comprise about 0.1 μm or less. In some embodiments, the length of the alternating sections may comprise about 1 μm or more. In some embodiments, the alternating sections may comprise about 0.3 μm in length. In some embodiments, the alternating sections may comprise about 0.6 μm in length.

[0098] In some embodiments, the alternating sections may comprise 10 base pairs or less. In some embodiments, the alternating sections may comprise 100 base pairs or more. In some embodiments, the alternating sections may comprise 10-100 base pairs. In some embodiments, the alternating sections may comprise 28 base pairs.

[0099] In some embodiments, the alternating compartments may contain 10 or fewer fluorophores. In some embodiments, the alternating compartments may contain 10 or more fluorophores. In some embodiments, the alternating compartments may contain 10 fluorophores to 1000 fluorophores. In some embodiments, the alternating compartments may contain 42 fluorophores, as shown in FIG. 10A. In some embodiments, the alternating compartments may contain 21 fluorophores, as shown in FIG. 11A.

[0100] Figure 10B shows a fluorescence image of a copy of the DNA polymer shown in Figure 10A, where the DNA has been stretched on a glass substrate according to some embodiments. Figure 10C shows a plot of fluorescence intensity versus distance (μm). Figure 10C shows a plot of 10 individual traces (e.g., fluorescence line profiles) extracted from the fluorescence image of the stretched DNA polymer. The bold line in Figure 10C represents the average of the individual traces.

[0101] Figure 11A shows a DNA polymer containing alternating segments, where according to some embodiments, each alternating segment is 0.6 kb, 0.3 μm long, and contains 21 fluorophores. Figure 11B shows a fluorescence image of the DNA polymer shown in Figure 11A. Figure 11C shows a plot of 10 individual traces (e.g., fluorescence line profiles) extracted from a fluorescence image of a stretched DNA polymer. The bold line in Figure 11C represents the average of the individual traces. EXAMPLES

[0102] Various examples of compositions, systems, and methods for polymer-based data storage are described herein. Examples of incorporation of chemically modifiable groups into polymers, methods for writing data, and methods for reading data are provided.

[0103] Example 1 Creating Data-Encodeable DNA In this example, the repeat sequence (GTA) n A DNA strand of is enzymatically constructed by rolling circle amplification. During polymerase extension, dGTP, dATP, and fluorescein-dUTP are supplied such that fluorescein is incorporated every third nucleotide. In a separate rolling circle reaction, (TAC) nA single stranded DNA having the sequence of is prepared by using polymerase-mediated incorporation of rhodamine-dUTP. Once a polymer strand of the desired length is produced, the two polymer strands are sequestered from the polymerase and unreacted dNTPs. The strands are then hybridized in a buffer of ionic strength that supports hybridization. The product is a number of long DNA duplexes containing the two dyes near each other on opposite strands, similar to the example depicted in Figure 3B.

[0104] Example 2 Data encoding by photobleaching of fluorophore clusters In this example, modified DNA molecules are constructed to contain side chains at thymidine residues, where the side chains have the photobleachable fluorophores fluorescein (green) and rhodamine (red) dyes linked to the amine groups of the thymine bases. Encoding is performed by passing the DNA through a plasmonic nanopore with an optical resolution of 5 nm. As the DNA passes through the pore, a pulse of light at a wavelength that excites the fluorescein is directed at the plasmonic structure, resulting in high energy focusing on a consecutive row of five of these bit modules. This causes bleaching of the fluorescein in clusters of five, resulting in a written "0" bit. As the DNA passes further, small segments of unbleached dye are left in between. As the DNA passes further, a pulse of light that excites the rhodamine is directed at the device, resulting in excitation and photobleaching of a cluster of five rhodamine dyes. This represents a "1" bit written as a cluster of dyes. Reading this DNA involves observing predominantly red signals, mixed green / red signals, and predominantly green signals, which represent the codes (from left to right) 0, space, and 1.

[0105] Example 3 Preparation of Cy-5-labeled DNA and localized photobleaching In this example, we demonstrate the stretching and writing of fluorophore (Cy-5) labeled synthetic DNA.

[0106] In this example, the following sequence (SEQ ID NO:1) was used:

[0107] [ka] [ka] [ka] [ka] [ka]

[0108] Preparation of DNA using plasmids DNA having the above sequence (SEQ ID NO:1) can be synthesized using a plasmid and DNA polymerase by replacing T with dye-labeled dUTP (such as Cy5-dUTP), thereby producing DNA with a labeled U, such as (wherein N can be A or C or G): ……NNNNNNNNNNUNNNUNNNUNNNUNNN…… It was prepared by bringing about

[0109] DNA cleanup The reaction mixture containing the prepared DNA was mixed with 100 μL of CleanNGS DNA&RNA Clean-Up Magnetic Beads, incubated at room temperature for 5 min, and left on the magnetic stand for 3 min. The clear liquid was aspirated and discarded. The magnetic beads were washed twice with 400 μL of 70% ethanol, incubated for 1 min each time. After the ethanol wash, the beads were air-dried for 1 min and then incubated on the rack with 50 μL of IDTE buffer for 5 min. The tube was then returned to the magnetic stand and left for 3 min. After all the magnetic beads had moved to the side, the clear liquid was transferred to a new 1.5 mL Eppendorf tube. The concentration of DNA was measured using a Qubit fluorometer and the integrity was confirmed using gel electrophoresis. Typically, 500-1000 ng of labeled DNA was recovered. An example of electrophoresis of labeled DNA can be seen in Figure 7, where lane 1 contains Cy5-labeled DNA and lane M contains Safegreen 1 kb DNA ladder.

[0110] Combing and stretching DNA Materials for the DNA combing protocol included: fiber comb instrument, genomic DNA extraction kit, stretching reservoir and vinylsilane-coated coverslips purchased from Genome Vision. Prolong Gold mounting solution was obtained from Thermofisher and YOYO-1 was obtained from Biotium.

[0111] In the DNA stretching protocol, 1 μL (20–50 ng) of labeled DNA was diluted in 2.4 mL of Buffer 6 from the genomic DNA extraction kit. The solution was warmed to 37°C for 5 min and then loaded into the reservoir. A vinylsilane-coated cover glass was incubated in the reservoir for 3 min, slowly removed, and the DNA was stretched onto the cover glass using a fiber comb instrument. The cover glass was heated to 125°C for 3 min and then loaded into Prolong with 1 μM YOYO-1. Slides were mounted with 10 μL of Gold mounting solution. Coverslips were sealed with nail polish.

[0112] DNA bleaching and imaging DNA stretched on a coverslip was imaged using a Cytiva OMX-SR microscope. The slide with the stretched DNA was loaded onto the microscope according to the manufacturer's instructions. Using a rectangular box perpendicular to the stretched DNA, a segment of Cy5-labeled DNA was bleached using a 633 nm laser for 25 ms.

[0113] The prepared DNA with Cy-5 labeling U was locally photobleached using light to write onto the DNA. The results of DNA imaging and bleaching are shown in Figures 8A-8C. The Y-axis shows the emission intensity and the X-axis is the distance (microns).

[0114] Figure 8A shows an image of Cy5-labeled DNA before bleaching, and Figure 8B shows images of Cy5-labeled DNA after sequential photobleaching. Figure 8C shows a plot showing two transverse profiles: before bleaching (1501) and after bleaching (1502). These show three distinct sites of bleaching, observable as dark spots of DNA and valleys in the intensity plot. The peak to valley distance is approximately 700 nm. From Figure 8C, a separation between the pre-bleaching state represented by the pre-bleaching profile (1501) and the post-bleaching state represented by the post-bleaching profile (1502) is clearly seen.

[0115] Example 4 Data encoding by photobleaching of fluorophore clusters In this example, a 40 kb region of double-stranded DNA was constructed to contain approximately 10,000 fluorescent dyes (StarRed, conjugated to deoxyuridine) repeated every 4 base pairs in the sequence, as shown in Figure 9A. The StarRed dye has an excitation maximum of approximately 638 nm and an emission maximum of approximately 655 nm. The DNA was then stretched onto glass to a length of approximately 16 μm using Genomic Vision's FiberComb Molecular Combing System, allowing the observation of single DNA molecules. The first image was acquired (labeled "Before"), as shown in Figure 9B. This stretched, writeable DNA polymer corresponds to a density of approximately 625 fluorophores per μm, and was monitored under a Cytiva OMX-SR microscope. A 640 nm laser was focused and used to photobleach a spot of approximately 1 μm in diameter on the DNA, thereby obtaining several "0" bits to binary encode the symbol "NA" (10011101000001). Bleaching was performed for 25 ms at each spot using 60 mW laser power. The laser light was automatically advanced from one position to the next. After bleaching, a second image was acquired (labeled "after") to read the written information, as shown in FIG. 9B. Ten traces (e.g., fluorescent line profiles) of the photobleached DNA are shown in FIG. 9C. A second example of DNA photobleaching was also performed to encode the symbol "IO" (1001001 1001110), as shown in FIG. 9D. In this way, approximately 625 dyes were used as a cluster to encode information for each binary "bit," encoding a string of digital information with consecutive bits. Note that the quantified fluorescence traces along the DNA were read individually for each DNA polymer, and the traces were averaged to allow for better accuracy in analyzing the string of encoded bits, as shown in Figures 9C and 9D.

[0116] Example 5 Data encoding and readout with prefabricated clusters of fluorophores In this example, a 6 kb region of double-stranded DNA was constructed to contain alternating 1.2 kb segments. As shown in FIG. 10A, each alternating segment contained only Cy5-linked deoxyuridine (total of 42 fluorophores per segment) or deoxyadenine, deoxycytosine, and deoxyguanine residues every 28 base pairs. The Cy5-deoxyuridine-containing segments were fluorescent, resulting in a "1" bit, while segments lacking Cy5 uridine remained dark, resulting in a "0" bit. To read this digital information within the polymer, the DNA was then stretched onto glass to a length of approximately 3 μm using Genomic Vision's FiberComb Molecular Combing System and monitored under a Cytiva OMX-SR microscope. Imaging revealed alternating light and dark strips, as shown in FIG. 10B, with each strip measuring 0.6 μm in length. A plot (thin line) and average (thick line) of ten traces (e.g., fluorescence intensity line profiles) of separate stretched DNA segments are shown in FIG. 10C. The plot displays the binary string "10101," confirming that a single DNA bit can be accurately read optically. In this example, a cluster of 42 dyes was used to encode a "1" bit, and a similar spacing with little or no fluorescence was used to encode a "0."

[0117] Similarly, a second DNA was constructed with alternating segments containing 0.6 kb and Cy5. In this example, segments alternated between segments containing 21 fluorophores and segments without fluorophores, as shown in FIG. 11A. In this case, light and dark strips were observed, as shown in FIG. 11B, and each strip was measured to be approximately 0.3 μm long. A plot of 10 traces (e.g., thin lines) and an average (e.g., thick lines) of separate stretched DNA segments is shown in FIG. 11C, confirming that the binary string "101010101" was encoded. This data confirms that a cluster of 21 fluorophores in a DNA polymer can be used to encode a single bit, and that the digital string can be read at the single molecule level.

Claims

1. 1. A method for encoding data onto a writeable polymer, comprising the steps of: providing a data-encodable nucleic acid polymer having a sequence of nucleotides with repeated repeats of one or more chemically-modifiable structures, each chemically-modifiable structure being attached onto the nucleic acid polymer, the one or more chemically-modifiable structures being capable of being modified to a second structural state by a pulse of light energy or redox energy; utilizing a data encoding device to selectively modify a subset of the one or more chemically modifiable structures along the nucleic acid polymer to the second structural state, thereby producing a data-encoded polymer defined by a plurality of modified structures, wherein the modified structures are arranged as clusters of repeating units; The length of each cluster is determined by the spatial resolution of the data encoding device, A method comprising:

2. The method of claim 1 , wherein the cluster comprises at least two sequentially modified structures.

3. The method of claim 1 , wherein the cluster comprises at least four consecutively modified structures.

4. The method of claim 1 , wherein the cluster comprises at least 21 consecutively modified structures.

5. The method of claim 1 , wherein the selectively modifying step comprises forming a single cluster at a time.

6. 2. The method of claim 1, wherein the selectively modifying step comprises simultaneously modifying a plurality of the chemically-modifiable structures to form clusters.

7. 2. The method of claim 1 , wherein the selectively modifying step comprises modifying a first subset of the one or more chemically-modifiable structures along the nucleic acid polymer to form a first cluster, leaving unmodified a second subset of the one or more chemically-modifiable structures positioned along the nucleic acid polymer after the first cluster in the 5' to 3' direction, and then modifying a third subset of the one or more chemically-modifiable structures along the nucleic acid polymer to form a third cluster positioned along the nucleic acid polymer after the second subset of the one or more chemically-modifiable structures in the 5' to 3' direction.

8. The method of claim 1 , wherein the cluster of repeating units comprises consecutively repeating units.

9. 9. The method of claim 8, wherein the cluster of consecutive repeating units comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, or 21 repeating units of a modified structure.

10. 9. The method of claim 8, wherein said cluster of consecutive repeating units comprises at least 21 repeating units.

11. 9. The method of claim 8, wherein said cluster of contiguous repeating units comprises at least 625 repeating units.

12. 9. The method of claim 8, wherein said cluster of contiguous repeating units comprises a repeating unit of at least about 0.6 kbp.

13. 9. The method of claim 8, wherein said cluster of contiguous repeating units comprises repeating units of at least about 1.2 kbp.

14. 9. The method of claim 8, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

15. 9. The method of claim 8, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

16. 9. The method of claim 8, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance on the order of at least 1 micron for the length of the polymer.

17. 2. The method of claim 1, wherein the cluster of repeating units is repeatedly repeated at least every 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 21, 24, 28, 32, 40, 80, 120 or 625 nucleotides.

18. 2. The method of claim 1, wherein said cluster of repeating units is repeatedly repeated at least every 21 units.

19. 2. The method of claim 1, wherein said cluster of repeating units is repeatedly repeated at least every 625 units.

20. 2. The method of claim 1, wherein said cluster of repeating units comprises a repeating unit of at least about 0.6 kbp.

21. 2. The method of claim 1, wherein said cluster of repeating units comprises a repeating unit of at least about 1.2 kbp.

22. 10. The method of claim 1, wherein the clusters of repeating units define a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

23. 10. The method of claim 1, wherein the clusters of repeating units define a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

24. 2. The method of claim 1, wherein the clusters of repeating units define a data bit resolution defined by a distance on the order of at least 1 micron for the length of the polymer.

25. the data encoding device comprises a nanopore, and the method further comprises: and further comprising passing the encodable nucleic acid polymer through the nanopore of the data encoding device, the nanopore comprising means for applying a pulse of light energy or redox energy to the subset of the one or more chemically modifiable structures to the second structural state. The method of claim 1.

26. 2. The method of claim 1, wherein each chemically-modifiable structure of the one or more chemically-modifiable structures comprises a caging group, a quencher, a photobleachable fluorophore, a photoconvertible fluorophore, or any combination thereof.

27. 2. The method of claim 1 , wherein a first chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a first wavelength of light.

28. 28. The method of claim 27, wherein a second chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a second wavelength of light.

29. 29. The method of claim 28, wherein the first chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at the second wavelength of light, and the second chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at the first wavelength of light.

30. 29. The method of claim 28, wherein the first chemically-modifiable structure and the second chemically-modifiable structure are attached to a repeated nucleotide structure.

31. 29. The method of claim 28, wherein the nucleic acid polymer is double stranded, the first chemically modifiable structure is attached to a repeated nucleotide structure on the top strand of the nucleic acid polymer, and the second chemically modifiable structure is attached to a repeated nucleotide structure on the bottom strand of the nucleic acid polymer.

32. 2. The method of claim 1, wherein the one or more chemically modifiable structures are repeatedly repeated at every second nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth nucleotide, every ninth nucleotide, every tenth nucleotide, every eleventh nucleotide, every twelfth nucleotide, every thirteenth nucleotide, every fourteenth nucleotide, every fifteenth nucleotide, every sixteenth nucleotide, every seventeenth nucleotide, every eighteenth nucleotide, every nineteenth nucleotide, every twentieth nucleotide, every twenty-first nucleotide, every twenty-second nucleotide, every twenty-third nucleotide, every twenty-fourth nucleotide, or every twenty-fifth nucleotide.

33. The method of claim 1, wherein the polymer comprises DNA, RNA, phosphorothioate DNA, glycerol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro-DNA 2'-O-methyl RNA, or locked nucleic acid (LNA).

34. 10. The method of claim 1, further comprising the step of decoding the encoded data of the selectively modified nucleic acid polymer.

35. 35. The method of claim 34, wherein the encoded data is decoded by passing the selectively modified nucleic acid polymer through a nanopore of a data decoding device, the nanopore of the data decoding device comprising means for detecting chemically modified structures within each cluster.

36. 35. The method of claim 34, wherein the data decoding device is the same device as the data encoding device.

37. 35. The method of claim 34, wherein the nanopore for decoding data is the same nanopore for encoding data.

38. 35. The method of claim 34, wherein the encoded data is decoded by stretching and imaging the selectively modified nucleic acid polymer.

39. The method of claim 1 , wherein the photo-modifiable group comprises a photo-removable leaving group that is not attached by a linker.

40. 2. The method of claim 1, wherein the photo-modifiable group comprises a photo-removable leaving group that is cleavable by light at a carbonyl bond, a thio (S) bond, or an NR2 bond.

41. 2. The method of claim 1, wherein the plurality of convertible nucleobases are converted from the first state to the second state by light of a wavelength of 325 nm, 360 nm, or 400 nm.

42. 2. The method of claim 1, wherein the plurality of convertible nucleobases are converted from the first state to the second state by light of a wavelength between 400 nm and 850 nm.

43. 2. The method of claim 1, wherein at least three convertible residues in the first state are between a data encoding segment of the polymer that includes a plurality of convertible residues in the second state.

44. The method of claim 1 , wherein at least three convertible residues in the first state are utilized for every nanometer of spatial resolution at which the data is encoded.

45. The method of claim 1 , further comprising a plurality of spacers between the data encoding segments of the polymer.

46. The method of claim 1 , wherein at least three spacer residues are utilized for every nanometer of spatial resolution at which the data is encoded.

47. The method of claim 1 , wherein the spacer moiety is a nucleobase that is not photomodifiable.

48. The method of claim 1 , wherein the convertible residue comprises a covalently attached monomeric unit.

49. 2. The method of claim 1, wherein the chemically modifiable structure comprises a photomodifiable group that includes a leaving group that is removable by light.

50. The method of claim 1 , wherein the convertible residues comprise covalently attached monomeric units linked consecutively along substantially the entire length of the polymer.

51. The method of claim 1 , wherein the convertible residues comprise covalently linked monomeric units and the data is encoded in the covalently attached monomeric units.

52. The method of claim 1 , wherein the convertible residues comprise covalently linked monomeric units and the data is encoded in the consecutive covalently attached monomeric units.

53. The method of claim 1 , wherein the polymer is defined by covalently linked monomeric units that do not hybridize in a side-by-side orientation.

54. The method of claim 1 , wherein the polymer is defined by covalently linked monomeric units, and the polymer does not hybridize in a side-by-side orientation with a second polymer that is not covalently linked.

55. The method of claim 1 further comprising forming a single-stranded polymer.

56. 10. The method of claim 1 , wherein the writable polymer is DNA, the method further comprising creating a second polymer that is complementary to the data-encoded polymer over approximately at least a portion of the data-encoded polymer.

57. The method of claim 1 , further comprising hybridizing at least a portion of the data-encoded polymer to the second polymer.

58. 10. The method of claim 1, further comprising hybridizing, in a 5' to 3' direction, at least a portion of the data encoded polymer to the second polymer.

59. The method of claim 1 , wherein the polymer comprises up to two consecutive covalently linked polymer molecules.

60. 53. The method of claim 52, wherein the two consecutive covalently linked polymer molecules form a DNA duplex.

61. 54. The method of claim 53, wherein the DNA duplex hybridizes in a 3' to 5' direction.

62. The method of claim 1 , wherein the repeatedly spaced convertible residues are located on a single covalently linked polymer.

63. 2. The method of claim 1, further comprising heating, cooling, reheating, or any combination thereof, the data-encoded polymer, wherein during the heating, cooling, reheating, or any combination thereof, the data-encoded polymer maintains its residue positions in approximately the 3' to 5' direction.

64. The method of claim 1 , wherein the chemically modifiable moiety is directly linked to one or more nucleobases.

65. The method of claim 1 , wherein the chemically modifiable moieties are repeatedly spaced along approximately the length of the polymer.

66. 59. The method of claim 58, wherein the repeat spacing along the length of the polymer is present every at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 2500, or at least 5000 spacer residues.

67. 2. The method of claim 1, wherein the selectively modifying step comprises removing a photoremovable leaving group for at least every 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 2500, or 5000 spacer residues.

68. 2. The method of claim 1, wherein the selectively modifying step comprises locally photocleaving a single photoremovable leaving group at a time.

69. 2. The method of claim 1, wherein there is no secondary structure attached to the polymer other than the chemically modifiable moiety directly linked to the one or more nucleobases.

70. The method of claim 1 , wherein the polymer does not have any data encoded therein when the plurality of convertible residues are in the first state.

71. The method of claim 1 , wherein the polymer is a writeable polymer configured to write data onto the polymer.

72. The method of claim 1 , wherein the polymer encodes data when at least a portion of the plurality of convertible residues are in the second state.

73. 2. The method of claim 1, wherein the selectively modifying step encodes data by cleaving at least a portion of the photoremovable leaving groups from the chemically-modifiable moiety to form the data-encoded polymer.

74. 2. The method of claim 1, wherein the selectively modifying step comprises reagent-free cleaving of the photo-removable leaving group from a nucleobase of the one or more nucleobases.

75. The method of claim 1 , wherein selectively modifying comprises translocating the polymer through a nanopore.

76. 2. The method of claim 1, wherein the selectively modifying step comprises translocating the polymer unidirectionally through the nanopore without translocating the polymer in a reverse direction through the nanopore.

77. The method of claim 1 , wherein the selectively modifying step comprises translocating the polymer through a nanopore at a continuous speed.

78. 2. The method of claim 1, wherein the selectively modifying step comprises writing data along the length of the polymer over a length of at least 1000, 2000, 3000, 4000, 5000, 10000, or 500000 residues.

79. 58. The method of claim 57, wherein said selectively modifying step does not include a step of oxidizing said one or more nucleobases.

80. The method of claim 1 , wherein the chemically modifiable moiety comprises a molecule comprising a plurality of distinct atoms.

81. The method of claim 1 , wherein the chemically modifiable moiety comprises a molecule that includes atoms other than oxygen (O).

82. The method of claim 1 , wherein the photoremovable leaving group comprises a molecule comprising multiple atoms.

83. The method of claim 1 , wherein the photoremovable leaving group comprises a molecule comprising a plurality of discrete atoms.

84. The method of claim 1 , wherein the photoremovable leaving group comprises a molecule containing an atom other than O.

85. 2. The method of claim 1, further comprising the step of reading the encoded data on the polymer by passing the writable polymer with data encoded thereon through a data reading device to identify whether each of the plurality of transformable residues that pass through the data reading device contains the photo-removable leaving group.

86. A data encoded polymer utilizing data encoding having a resolution that results in clusters of repeatedly spaced modified structures, a nucleic acid polymer having a sequence of nucleotides in which one or more chemically modifiable structures are repeatedly repeated; the nucleic acid polymer comprises clusters of repeating units of a modified structure, the clusters being repeated repeatedly along the nucleic acid; the modified structure is chemically modified from a first structural state to a second structural state by a pulse of light or redox energy; The plurality of clusters defines encoded data determined by the modified structures within each cluster.

87. 87. The polymer of claim 86, wherein the cluster of repeating units comprises consecutive repeating units of the modified structure in the second structural state.

88. 87. The polymer of claim 86, wherein the cluster of consecutive repeating units comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units of the modified structure.

89. 87. The polymer of claim 86, wherein the cluster of consecutive repeating units comprises at least 21 repeating units of the modified structure.

90. 87. The polymer of claim 86, wherein the cluster of consecutive repeating units comprises at least 625 repeating units of the modified structure.

91. 87. The polymer of claim 86, wherein the cluster of consecutive repeating units comprises at least about 0.6 kbp of repeating units of the modified structure.

92. 87. The polymer of claim 86, wherein the cluster of consecutive repeating units comprises at least about 1.2 kbp of repeating units of the modified structure.

93. 87. The polymer of claim 86, wherein said clusters of consecutive repeating units define a data bit resolution defined by a distance of at least 0.3 microns over the length of said polymer.

94. 87. The polymer of claim 86, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

95. 87. The polymer of claim 86, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer.

96. 87. The polymer of claim 86, wherein the cluster of repeating units is repeatedly repeated at least every 2, 3, 4, 5, 6, 7, 8, 9 or 10 units.

97. 87. The polymer of claim 86, wherein the cluster of repeating units is repeatedly repeated at least every 21 units.

98. 87. The polymer of claim 86, wherein the cluster of repeating units is repeatedly repeated at least every 625 units.

99. 87. The polymer of claim 86, wherein the cluster of repeating units comprises at least about 0.6 kbp of repeating units.

100. 87. The polymer of claim 86, wherein the cluster of repeating units comprises at least about 1.2 kbp of repeating units.

101. 87. The polymer of claim 86, wherein the clusters of repeating units define a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

102. 87. The polymer of claim 86, wherein the clusters of repeating units define a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

103. 87. The polymer of claim 86, wherein the clusters of repeating units define a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer.

104. 87. The polymer of claim 86, wherein the chemically modifiable structure comprises a photomodifiable group that includes a leaving group that is removable by light.

105. The polymer of claim 104, wherein the photomodifiable group comprises a photoremovable leaving group that is not attached by a linker.

106. The polymer of claim 104, wherein the photomodifiable group comprises a photoremovable leaving group that is cleavable by light at a carbonyl bond, a thio (S) bond, or an NR2 bond.

107. 105. The polymer of claim 104, wherein said plurality of convertible nucleobases are converted from said first state to said second state by light of a wavelength of 325 nm, 360 nm, or 400 nm.

108. 105. The polymer of claim 104, wherein said plurality of convertible nucleobases are converted from said first state to said second state by light of a wavelength between 400 nm and 850 nm.

109. 87. The polymer of claim 86, wherein at least three convertible residues in the first state are between a data encoding segment of the polymer that includes a plurality of convertible residues in the second state.

110. 87. The polymer of claim 86, wherein at least three convertible residues in the first state are utilized for every nanometer of spatial resolution along which the data is encoded.

111. 90. The polymer of claim 86, further comprising a plurality of spacers between the data encoding segments of the polymer.

112. 87. The polymer of claim 86, wherein at least three spacer residues are utilized for every nanometer of spatial resolution at which the data is encoded.

113. 87. The polymer of claim 86, wherein the spacer moiety is a nucleobase that is not photomodifiable.

114. 87. The polymer of claim 86, wherein the convertible moiety comprises a covalently attached monomeric unit.

115. 87. The polymer of claim 86, wherein the convertible residue comprises covalently linked monomeric units that are linked consecutively along substantially the entire length of the polymer.

116. 87. The polymer of claim 86, wherein the convertible residues comprise covalently linked monomeric units and the data is encoded in the covalently linked monomeric units.

117. 87. The polymer of claim 86, wherein the convertible residue comprises covalently linked monomeric units and the data is encoded in consecutive covalently linked monomeric units.

118. 87. The polymer of claim 86, defined by covalently linked monomeric units that do not hybridize in the side-by-side orientation.

119. 87. The polymer of claim 86, defined by covalently linked monomeric units, wherein the polymer will not hybridize in the side-by-side direction to a second polymer that is not covalently linked.

120. 87. The polymer of claim 86, comprising a single molecule that forms a single-stranded polymer.

121. 87. The polymer of claim 86, defined by up to two DNA polymer molecules.

122. 87. The polymer of claim 86, defined by up to two hybridized DNA polymer molecules.

123. 87. The polymer of claim 86, defined by up to two hybridized DNA polymer molecules hybridized in a 5' to 3' orientation.

124. 87. The polymer of claim 86, comprising up to two contiguous covalently linked polymer molecules.

125. 125. The polymer of claim 124, wherein the two consecutive covalently linked polymer molecules form a DNA duplex.

126. 126. The polymer of claim 125, wherein the DNA duplex hybridizes in the 3' to 5' direction.

127. 87. The polymer of claim 86, wherein the repeatedly spaced convertible residues are located on a single covalently linked polymer.

128. 87. The polymer of claim 86, which maintains its residue positions in approximately the 3' to 5' direction when heated, cooled, reheated, or any combination thereof.

129. 87. The polymer of claim 86, which is not bound to any other polymer by hybridization.

130. 87. The polymer of claim 86, wherein the polymer comprises one or more nucleobases and the chemically modifiable moiety is directly linked to the one or more nucleobases.

131. 87. The polymer of claim 86, wherein the convertible residues that comprise the chemically modifiable moiety are repeatedly spaced approximately along the length of the polymer.

132. 132. The polymer of claim 131, wherein the repeat spacing along the length of the polymer is present every at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 2500, or at least 5000 spacer residues.

133. 87. The polymer of claim 86, configured for localized photocleavage of a single removable leaving group at a time.

134. 87. The polymer of claim 86, wherein there is no secondary structure attached to the polymer other than the chemically modifiable moiety directly linked to the one or more nucleobases.

135. 87. The polymer of claim 86, wherein when said plurality of convertible residues are in said first state, no data is encoded.

136. 87. The polymer of claim 86, which is a writeable polymer configured to write data onto the polymer.

137. 87. The polymer of claim 86, wherein data is encoded when at least a portion of said plurality of convertible residues are in said second state.

138. 105. The polymer of claim 104, configured to encode data by cleaving at least a portion of the photo-removable leaving groups from the polymer.

139. 139. The polymer of claim 138, wherein the chemically modifiable moiety is configured to be cleaved by the one or more nucleobases without the use of chemical reagents.

140. 87. The polymer of claim 86, configured to translocate unidirectionally through the nanopore.

141. 87. The polymer of claim 86, configured to translocate in one direction through the nanopore without translocating in the reverse direction through the nanopore.

142. 87. The polymer of claim 86, which is at least 1000, 2000, 3000, 4000, 5000, 10000, or 500000 residues in length.

143. 87. The polymer of claim 86, wherein the polymer in the second state does not comprise an oxidized nucleobase.

144. 87. The polymer of claim 86, wherein the second state is a written form that does not include oxidized nucleobases.

145. 87. The polymer of claim 86, wherein the chemically modifiable moiety comprises a chemical residue comprising a plurality of atoms.

146. 87. The polymer of claim 86, wherein the polymer in the second state does not include the chemically modifiable moiety.

147. 87. The polymer of claim 86, wherein the polymer in the second state does not include the chemical moiety that comprises the plurality of atoms.

148. 87. The polymer of claim 86, wherein the chemically modifiable moiety comprises a molecule that includes an atom other than O (oxygen).

149. 105. The polymer of claim 104, wherein the photoremovable leaving group comprises a molecule comprising multiple atoms.

150. The polymer of claim 104, wherein the photoremovable leaving group comprises a molecule comprising a plurality of discrete atoms.

151. The polymer of claim 104, wherein the photoremovable leaving group comprises a molecule that includes an atom other than O.

152. 105. The polymer of claim 104, wherein the first structural state of each modified structure of the plurality of modified structures comprises one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore.

153. 2. The polymer of claim 1, wherein the modified structures of a first cluster are one or more photobleached fluorophores that have been photobleached by a pulse of light energy at a first wavelength of light.

154. 2. The polymer of claim 1, wherein the modified structures of the second cluster are one or more photobleached fluorophores that have been photobleached by a pulse of light energy at a second wavelength of light.

155. 155. The polymer of claim 154, wherein the first cluster further comprises one or more photobleachable fluorophores that are photobleachable at the second wavelength of light but are not photobleached, and the second cluster further comprises one or more photobleachable fluorophores that are photobleachable at the first wavelength of light but are not photobleached.

156. The polymer of claim 155, wherein each photobleachable fluorophore in the first cluster that is photobleachable by the first wavelength is photobleached by a pulse of light energy at the first wavelength of light, and each photobleachable fluorophore in the second cluster that is photobleachable by the second wavelength is photobleached by a pulse of light energy at the second wavelength of light.

157. 156. The polymer of claim 155, wherein the modified structure and the one or more photobleachable fluorophores of the first cluster are attached to a repeated nucleotide structure.

158. 156. The polymer of claim 155, wherein the nucleic acid is double-stranded, the modified structure of the first cluster is attached to a repeated nucleotide structure on the top strand of the nucleic acid polymer, and the one or more photobleachable fluorophores of the first cluster are attached to a repeated nucleotide structure on the bottom strand of the nucleic acid polymer.

159. 87. The polymer of claim 86, wherein the length of each cluster of the plurality of clusters is equal to the spatial resolution of the pulse of light or redox energy used to modify the plurality of chemically modified structures from the first structural state to the second structural state.

160. 87. The polymer of claim 86, wherein the nucleic acid polymer comprises a plurality of spaces between each cluster of the plurality of clusters.

161. 87. The polymer of claim 86, wherein each interval of the plurality of intervals comprises one or more chemically modifiable structures that are not modified by a pulse of light or redox energy.

162. 87. The polymer of claim 86, wherein the one or more modified structures are repeatedly repeated at every second nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth nucleotide, every ninth nucleotide, every tenth nucleotide, every eleventh nucleotide, every twelfth nucleotide, every thirteenth nucleotide, every fourteenth nucleotide, every fifteenth nucleotide, every sixteenth nucleotide, every seventeenth nucleotide, every eighteenth nucleotide, every nineteenth nucleotide, every twentieth nucleotide, every twenty-first nucleotide, every twenty-second nucleotide, every twenty-third nucleotide, every twenty-fourth nucleotide, or every twenty-fifth nucleotide.

163. 87. The polymer of claim 86, comprising DNA, RNA, phosphorothioate DNA, glycerol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro-DNA 2'-O-methyl RNA, or locked nucleic acid (LNA).

164. 1. A data-encodeable polymer for encoding data using repeatedly spaced chemically modifiable structures, comprising: a nucleic acid polymer having a sequence of nucleotides in which one or more chemically modifiable structures are repeatedly repeated; each chemically-modifiable structure of the one or more chemically-modifiable structures is attached onto the nucleic acid polymer; A polymer, wherein the one or more chemically modifiable structures are capable of being modified from a first structural state to a second structural state in a cluster of a plurality of chemically modified structures by a pulse of light energy or redox energy.

165. 165. The polymer of claim 164, wherein the cluster of repeating units comprises consecutive repeating units.

166. 166. The polymer of claim 165, wherein the cluster of consecutive repeating units comprises at least 4, 8, 10, 12, 16, or 20 repeating units.

167. 166. The polymer of claim 165, wherein the cluster of consecutive repeating units comprises at least 21 repeating units.

168. 166. The polymer of claim 165, wherein the cluster of consecutive repeating units comprises at least 625 repeating units.

169. 166. The polymer of claim 165, wherein the cluster of consecutive repeating units comprises at least about 0.6 kbp of repeating units.

170. 166. The polymer of claim 165, wherein the cluster of consecutive repeating units comprises at least about 1.2 kbp of repeating units.

171. 166. The polymer of claim 165, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

172. 166. The polymer of claim 165, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

173. 166. The polymer of claim 165, wherein the clusters of consecutive repeating units define a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer.

174. 165. The polymer of claim 164, wherein the cluster of repeating units is repeated at least every 10 units.

175. 165. The polymer of claim 164, wherein the cluster of repeating units is repeatedly repeated at least every 21 units.

176. 165. The polymer of claim 164, wherein the cluster of repeating units is repeatedly repeated at least every 625 units.

177. 165. The polymer of claim 164, wherein the cluster of repeating units comprises at least about 0.6 kbp of repeating units.

178. 165. The polymer of claim 164, wherein the cluster of repeating units comprises at least about 1.2 kbp of repeating units.

179. 165. The polymer of claim 164, wherein the clusters of repeating units define a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

180. 165. The polymer of claim 164, wherein the clusters of repeating units define a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

181. 165. The polymer of claim 164, wherein the clusters of repeating units define a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer.

182. 165. The polymer of claim 164, wherein each chemically-modifiable structure of the one or more chemically-modifiable structures comprises one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore.

183. The polymer of claim 164, wherein a first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a first wavelength of light.

184. 184. The polymer of claim 183, wherein the first chemically modifiable structure is attached to a repeated nucleotide structure.

185. The polymer of claim 183, wherein a second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a second wavelength of light.

186. The polymer of claim 185, wherein the first chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at the second wavelength of light, and the second chemically modifiable structure of the one or more chemically modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at the first wavelength of light.

187. 186. The polymer of claim 185, wherein the first chemically modifiable structure and the second chemically modifiable structure are attached to a repeated nucleotide structure.

188. The polymer of claim 185, wherein the nucleic acid polymer is double-stranded, the first chemically-modifiable structure is attached to a repeated nucleotide structure on the top strand of the nucleic acid polymer, and the second chemically-modifiable structure is attached to a repeated nucleotide structure on the bottom strand of the nucleic acid polymer.

189. 165. The polymer of claim 164, wherein the one or more chemically modifiable structures comprise a photoconvertible fluorophore that exists in a first structural state having a first emission wavelength and is convertible to a second structural state having a second emission wavelength by a pulse of light or redox energy.

190. The polymer of claim 189, wherein the conversion of the photoconvertible fluorophore from the first structural state to the second structural state is by a pulse of light at a first wavelength, and the photoconvertible fluorophore is capable of being converted to a third structural state having a third emission wavelength by a pulse of light at a second wavelength.

191. The polymer of claim 164, wherein the one or more chemically modifiable structures are repeatedly repeated at every second nucleotide, every third nucleotide, every fourth nucleotide, every fifth nucleotide, every sixth nucleotide, every seventh nucleotide, every eighth nucleotide, every ninth nucleotide, every tenth nucleotide, every eleventh nucleotide, every twelfth nucleotide, every thirteenth nucleotide, every fourteenth nucleotide, every fifteenth nucleotide, every sixteenth nucleotide, every seventeenth nucleotide, every eighteenth nucleotide, every nineteenth nucleotide, every twentieth nucleotide, every twenty-first nucleotide, every twenty-second nucleotide, every twenty-third nucleotide, every twenty-fourth nucleotide, or every twenty-fifth nucleotide.

192. 165. The polymer of claim 164, comprising DNA, RNA, phosphorothioate DNA, glycerol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro-DNA 2'-O-methyl RNA, or locked nucleic acid (LNA).

193. The polymer of claim 164, wherein the chemically modifiable structure comprises a photomodifiable group that includes a leaving group that is removable by light.

194. The polymer of claim 164, wherein the photomodifiable group comprises a photoremovable leaving group that is not attached by a linker.

195. The polymer of claim 164, wherein the photo-modifiable group comprises a photo-removable leaving group that is cleavable by light at a carbonyl bond, a thio (S) bond, or an NR2 bond.

196. 165. The polymer of claim 164, wherein said plurality of convertible nucleobases are converted from said first state to said second state by light of a wavelength of 325 nm, 360 nm, or 400 nm.

197. 165. The polymer of claim 164, wherein said plurality of convertible nucleobases are converted from said first state to said second state by light of a wavelength between 400 nm and 850 nm.

198. 165. The polymer of claim 164, wherein at least three convertible residues in the first state are between a data encoding segment of the polymer that includes a plurality of convertible residues in the second state.

199. 165. The polymer of claim 164, wherein at least three convertible residues in the first state are utilized for every nanometer of spatial resolution at which the data is encoded.

200. 165. The polymer of claim 164, further comprising a plurality of spacers between the data encoding segments of the polymer.

201. 165. The polymer of claim 164, wherein at least three spacer residues are utilized for every nanometer of spatial resolution at which the data is encoded.

202. 165. The polymer of claim 164, wherein the spacer moiety is a nucleobase that is not photomodifiable.

203. 165. The polymer of claim 164, wherein the convertible residue comprises a covalently attached monomeric unit.

204. 165. The polymer of claim 164, wherein the convertible residue comprises covalently linked monomeric units that are linked consecutively along substantially the entire length of the polymer.

205. 165. The polymer of claim 164, wherein the convertible residues comprise covalently linked monomeric units and the data is encoded in the covalently linked monomeric units.

206. 165. The polymer of claim 164, wherein the convertible residue comprises covalently linked monomeric units and the data is encoded in consecutive covalently linked monomeric units.

207. 165. The polymer of claim 164, which is defined by covalently linked monomeric units and which do not hybridize in the lateral direction.

208. 165. The polymer of claim 164, defined by covalently linked monomeric units, wherein the polymer will not hybridize in the side-by-side direction to a second polymer that is not covalently linked.

209. 165. The polymer of claim 164, comprising a single molecule that forms a single-stranded polymer.

210. 165. The polymer of claim 164, defined by up to two DNA polymer molecules.

211. 165. The polymer of claim 164, defined by up to two hybridized DNA polymer molecules.

212. 165. The polymer of claim 164, defined by up to two hybridized DNA polymer molecules hybridized in a 5' to 3' orientation.

213. 165. The polymer of claim 164, comprising up to two consecutive covalently linked polymer molecules.

214. 165. The polymer of claim 164, wherein the two consecutive covalently linked polymer molecules form a DNA duplex.

215. The polymer of claim 214, wherein the DNA duplex hybridizes in the 3' to 5' direction.

216. 165. The polymer of claim 164, wherein the repeatedly spaced convertible residues are located on a single polymer that is covalently linked.

217. 165. The polymer of claim 164, which maintains its residue positions in approximately the 3' to 5' direction when heated, cooled, reheated, or any combination thereof.

218. 165. The polymer of claim 164, which is not bound to any other polymer by hybridization.

219. 165. The polymer of claim 164, wherein the polymer comprises one or more nucleobases, and the chemically modifiable moiety is directly linked to the one or more nucleobases.

220. 165. The polymer of claim 164, wherein the convertible residues that comprise the chemically modifiable moiety are repeatedly spaced approximately along the length of the polymer.

221. 165. The polymer of claim 164, wherein the repeat spacing along the length of the polymer is present every at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 2500, or at least 5000 spacer residues.

222. 165. The polymer of claim 164, configured for localized photocleavage of a single removable leaving group at a time.

223. 165. The polymer of claim 164, wherein there is no secondary structure attached to the polymer other than the chemically modifiable moiety directly linked to the one or more nucleobases.

224. 165. The polymer of claim 164, wherein when the plurality of convertible residues are in the first state, no data is encoded.

225. 165. The polymer of claim 164, which is a writeable polymer configured to write data onto the polymer.

226. 165. The polymer of claim 164, wherein data is encoded when at least a portion of said plurality of convertible residues are in said second state.

227. The polymer of claim 193, configured to encode data by cleaving at least a portion of the photoremovable leaving groups from the polymer.

228. 165. The polymer of claim 164, wherein the chemically modifiable moiety is configured to be cleaved by the one or more nucleobases without the use of chemical reagents.

229. The polymer of claim 164, configured to translocate unidirectionally through the nanopore.

230. 165. The polymer of claim 164, configured to translocate in one direction through the nanopore without translocating in the reverse direction through the nanopore.

231. 165. The polymer of claim 164, which is at least 1000, 2000, 3000, 4000, 5000, 10000, or 500000 residues in length.

232. 165. The polymer of claim 164, wherein the polymer in the second state does not contain any oxidized nucleobases.

233. 165. The polymer of claim 164, wherein the second state is a written form that does not include oxidized nucleobases.

234. The polymer of claim 164, wherein the chemically modifiable moiety comprises a chemical residue comprising a plurality of atoms.

235. 165. The polymer of claim 164, wherein the polymer in the second state does not include the chemically modifiable moiety.

236. 165. The polymer of claim 164, wherein the polymer in the second state does not include the chemical moiety comprising the plurality of atoms.

237. The polymer of claim 164, wherein the chemically modifiable moiety comprises a molecule that includes an atom other than O (oxygen).

238. The polymer of claim 193, wherein the photoremovable leaving group comprises a molecule comprising multiple atoms.

239. The polymer of claim 193, wherein the photoremovable leaving group comprises a molecule comprising a plurality of discrete atoms.

240. The polymer of claim 193, wherein the photoremovable leaving group comprises a molecule containing an atom other than O.

241. The polymer of claim 164, configured to encode data in clusters of chemically modified structures converted from the chemically modifiable structures.

242. 1. A data-encodeable polymer for encoding data using repeatedly spaced chemically modifiable structures, comprising: comprising a polymer having a sequence of monomers in which one or more chemically modifiable structures are repeatedly repeated in clusters of repeating units; each chemically-modifiable structure of the one or more chemically-modifiable structures is attached onto the nucleic acid polymer via a monomer; The method, wherein said one or more chemically modifiable structures are capable of being modified by a pulse of light energy or redox energy.

243. 243. The method of claim 242, wherein said cluster of repeating units comprises contiguous repeating units.

244. 244. The method of claim 243, wherein said cluster of contiguous repeating units comprises at least 10 repeating units.

245. 244. The method of claim 243, wherein said cluster of contiguous repeating units comprises at least 21 repeating units.

246. 244. The method of claim 243, wherein said cluster of contiguous repeating units comprises at least 625 repeating units.

247. 244. The method of claim 243, wherein said cluster of contiguous repeating units comprises a repeating unit of at least about 0.6 kbp.

248. 244. The method of claim 243, wherein said cluster of contiguous repeating units comprises a repeating unit of at least about 1.2 kbp.

249. 244. The method of claim 243, wherein the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

250. 244. The method of claim 243, wherein the cluster of consecutive repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

251. 244. The method of claim 243, wherein the cluster of consecutive repeating units defines a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer.

252. 243. The method of claim 242, wherein said cluster of repeating units is repeatedly repeated at least every 10 units.

253. 243. The method of claim 242, wherein said cluster of repeating units is repeatedly repeated at least every 21 units.

254. 243. The method of claim 242, wherein said cluster of repeating units is repeatedly repeated at least every 625 units.

255. 243. The method of claim 242, wherein said cluster of repeating units comprises a repeating unit of at least about 0.6 kbp.

256. 243. The method of claim 242, wherein said cluster of repeating units comprises a repeating unit of at least about 1.2 kbp.

257. 243. The method of claim 242, wherein the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.3 microns over the length of the polymer.

258. 243. The method of claim 242, wherein the cluster of repeating units defines a data bit resolution defined by a distance of at least 0.6 microns over the length of the polymer.

259. 243. The method of claim 242, wherein the clusters of repeating units define a data bit resolution defined by a distance on the order of at least 1 micron over the length of the polymer.

260. 243. The method of claim 242, wherein each chemically-modifiable structure of the one or more chemically-modifiable structures comprises one of the following: a caging group, a quencher, a photobleachable fluorophore, or a photoconvertible fluorophore.

261. 243. The method of claim 242, wherein a first chemically-modifiable structure of said one or more chemically-modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a first wavelength of light.

262. 262. The method of claim 261, wherein a second chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is photobleachable by a pulse of light energy at a second wavelength of light.

263. The method of claim 261, wherein the first chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at the second wavelength of light, and the second chemically-modifiable structure of the one or more chemically-modifiable structures is a photobleachable fluorophore that is not photobleachable by a pulse of light energy at the first wavelength of light.

264. The polymer described in claim 261, wherein the one or more chemically modifiable structures comprise a photoconvertible fluorophore that exists in a first structural state having a first emission wavelength and is convertible to a second structural state having a second emission wavelength by a pulse of light energy or redox energy.

265. 243. The method of claim 242, wherein the conversion of the photo-convertible fluorophore from the first structural state to the second structural state is by a pulse of light at a first wavelength, and the photo-convertible fluorophore is capable of being converted to a third structural state having a third emission wavelength by a pulse of light at a second wavelength.

266. 243. The method of claim 242, wherein the one or more chemically modifiable structures are repeatedly repeated as follows: every second monomer, every third monomer, every fourth monomer, every fifth monomer, every sixth monomer, every seventh monomer, every eighth monomer, every ninth monomer, every tenth monomer, every eleventh monomer, every twelfth monomer, every thirteenth monomer, every fourteenth monomer, every fifteenth monomer, every sixteenth monomer, every seventeenth monomer, every eighteenth monomer, every nineteenth monomer, every twentieth monomer, every twenty-first monomer, every twenty-second monomer, every twenty-third monomer, every twenty-fourth monomer, or every twenty-fifth monomer.

267. 243. The method of claim 242, wherein the polymer is an inorganic polymer.

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