Abstract: Sequential assembly of oligonucleotide hairpins is used to create oligonucleotides with specific sequences. Each oligonucleotide hairpin includes a payload region containing one or more nucleotides that are added to the end of an anchor strand. Overhang regions on the oligonucleotide hairpins hybridize to anchor strands attached to a substrate. The hybridized oligonucleotide hairpins are covalently attached to the anchor strands by the activity of ligase. The oligonucleotide hairpins include an enzyme cleavage region which, when cleaved, separates the payload region from the remainder of the oligonucleotide hairpin. The oligonucleotide hairpin is separated from the anchor strand by denaturation and washed away. This process is repeated with additional oligonucleotide hairpins to add additional nucleotides to the ends of the anchor strands. A microelectrode array may be used to control the location of hybridization and create multiple oligonucleotides in parallel.

Abstract: Synthetic molecular tags are placed on an item at various points in a supply chain to create a molecular record of movement through the supply chain. Associations between each unique synthetic molecular tag and individual locations in the supply chain are stored in an electronic record which may be maintained in the cloud. The synthetic molecular tags are collected from the item and sequenced to determine movement of the item through the supply chain by reference to the electronic record. The synthetic molecular tags can be used for identifying recalled items based on locations in the supply chain associated with a recall. The synthetic molecular tags may be polynucleotides such as deoxyribose nucleic acid (DNA). The item may be any type of item including food.

Abstract: Fluorophores are used during the synthesis of oligonucleotides to achieve real-time quality control of the synthesis process. Fluorescence may indicate successful addition of individual nucleotides to a growing oligonucleotide strand or removal of a blocking group. The oligonucleotides may be created by enzymatic synthesis using terminal deoxynucleotidyl transferase (TdT). The synthesis is performed on an addressable array so that oligonucleotides with different sequences are created in parallel on different regions of the array. The oligonucleotide sequences are predetermined and the locations of synthesis on the array are controlled. Observed fluorescence is compared to expected locations of fluorescence as determined by the oligonucleotide sequences and the arrangement on the array. Thus, the fidelity of oligonucleotide synthesis is checked as synthesis proceeds. If a variation is found, a mitigating action is taken such as repeating addition of a species of nucleotide or repeating a deblocking step.

Abstract: Molecular anti-counterfeiting taggants are made from a plurality of synthetic polynucleotides that collectively encode a bit sequence using the sequences and hybridization states of the polynucleotides. The polynucleotide taggant is placed on an item as a molecular identifier of authenticity. The bit sequence encoded by the polynucleotide taggant is read out using a substrate which has bound polynucleotides complexes that hybridize with the synthetic polynucleotides in the polynucleotide taggant. A detectable signal is present where hybridization occurs. To prevent a bad actor from reverse engineering and creating a copy of the polynucleotide taggant using the results of hybridization to the substrate, multiple versions of the substrate are created. Each version hybridizes to different subsets of the synthetic polynucleotides in the polynucleotide taggant. A detectable pattern on the substrate that is present when exposed to the polynucleotide taggant is used for validating authenticity of the item.

Abstract: A data storage medium is disclosed comprising a two-dimensional (2D) support structure onto which artificially synthesized DNA molecules encoding digital information are placed and then covered with a protective layer. The 2D support structure is formed from a material such as metal foil, glass, or plastic. The 2D support structure may be functionalized with positively charged molecules to improve DNA adhesion. The DNA is protected from degradation by encapsulation in a protective layer of a non-reactive material such as silica or a thin layer of metal. A process for storing DNA on 2D support structures is also disclosed. Correlation of specific DNA molecules with a physical storage location on a 2D support structure provides geometric addressability for selective access to specific digital information.

Abstract: A multiplex assay for nucleic acid detection includes a substrate, a sample, and a fluorophore-labeled oligonucleotide. The substrate has a plurality of physically separated assay locations, each of which includes a nucleotide-targeting enzyme configured to cleave nucleic acids, a guide ribonucleic acid (gRNA), and a quencher-labeled oligonucleotide. A portion of the sample is distributed to each assay location. The gRNA recognizes target nucleic acid in the sample, thereby activating the nucleotide-targeting enzyme to cleave nucleic acids, including the quencher-labeled oligonucleotide. The fluorophore-labeled oligonucleotide is subsequently added to each assay location, which facilitates identification of a presence of the target nucleic acid in the sample via detection of unquenched light emitted by the fluorophore in one or more of the plurality of assay locations.

Abstract: Polynucleotides such as DNA are stored inside vesicles formed from self-assembling membranes. The vesicles may be protocells, liposomes, micelles, colloidosomes, proteinosomes, or coacervates. The vesicles may include surface functionalization to improve polynucleotide encapsulation and/or to bind polynucleotides having specific sequences. Encapsulation in vesicles provides protection for the polynucleotides. Additional protection is provided by addition of one or more stabilizers. The stabilizer may be nucleic-acid stabilizers that stabilize the polynucleotides or may be a protective structural layer around the vesicles such as a layer of silica. A process for stably storing polynucleotides in vesicles and a process for recovering stored polynucleotides from vesicles are both disclosed. The polynucleotides may be used for storage of digital information.

Abstract: Synthetic molecular tags are placed on an item at various points in a supply chain to create a molecular record of movement through the supply chain. Associations between each unique synthetic molecular tag and individual locations in the supply chain are stored in an electronic record which may be maintained in the cloud. The synthetic molecular tags are collected from the item and sequenced to determine movement of the item through the supply chain by reference to the electronic record. The synthetic molecular tags can be used for identifying recalled items based on locations in the supply chain associated with a recall. The synthetic molecular tags may be polynucleotides such as deoxyribose nucleic acid (DNA). The item may be any type of item including food.

Abstract: Recycling information is associated with objects through the use of molecular tags. The recycling information may describe the type of material that the object is made from as well as provide instructions for recycling. The molecular tags may be polynucleotides or other types of molecules including inorganic molecules. The molecular tags may be embedded within the object or attached to the surface of the object. At the end of the object's life, the molecular tags are read and the recycling information is used to appropriately recycle the object.

Abstract: A data storage medium is disclosed comprising a substrate covered with alternating layers of a polycationic molecule and artificially synthesized DNA molecules encoding digital information. The magnetic substrate may be a metallic nanoparticle formed from a metal such as iron or cobalt. The polycationic molecule may be polyethyleneimine (PEI). The DNA is protected from degradation by encapsulation in silica. A process for stably storing DNA is also disclosed. Stored DNA may be freed from the silica for sequencing or other analysis by washing the silica-coated DNA with a buffered hydrogen fluoride solution. Storage densities of more than 7% DNA by weight are achieved on nanoparticles.

Abstract: This disclosure describes an efficient method to copy all polynucleotides encoding digital data of digital files in a polynucleotide storage container while maintaining random access capabilities over a collection of files or data items in the container. The disclosure further describes a process whereby random-access and sequencing of the polynucleotides are combined in a single step.

Abstract: Polynucleotides such as DNA are stored inside vesicles formed from self-assembling membranes. The vesicles may be protocells, liposomes, micelles, colloidosomes, proteinosomes, or coacervates. The vesicles may include surface functionalization to improve polynucleotide encapsulation and/or to bind polynucleotides having specific sequences. Encapsulation in vesicles provides protection for the polynucleotides. Additional protection is provided by addition of one or more stabilizers. The stabilizer may be nucleic-acid stabilizers that stabilize the polynucleotides or may be a protective structural layer around the vesicles such as a layer of silica. A process for stably storing polynucleotides in vesicles and a process for recovering stored polynucleotides from vesicles are both disclosed. The polynucleotides may be used for storage of digital information.

Abstract: A multiplex assay for nucleic acid detection includes a substrate, a sample, and a fluorophore-labeled oligonucleotide. The substrate has a plurality of physically separated assay locations, each of which includes a nucleotide-targeting enzyme configured to cleave nucleic acids, a guide ribonucleic acid (gRNA), and a quencher-labeled oligonucleotide. A portion of the sample is distributed to each assay location. The gRNA recognizes target nucleic acid in the sample, thereby activating the nucleotide-targeting enzyme to cleave nucleic acids, including the quencher-labeled oligonucleotide. The fluorophore-labeled oligonucleotide is subsequently added to each assay location, which facilitates identification of a presence of the target nucleic acid in the sample via detection of unquenched light emitted by the fluorophore in one or more of the plurality of assay locations.

Abstract: Multiplex similarity search can be performed in a DNA data storage context. The described technologies can support a plurality of different DNA data storage queries in a single query run. A linking strand can be used to connect a query to its matching data element. After the query finds a matching data element, a result strand can be sequenced to the reveal the matching data element as well as which of the queries resulted in the match. Thus, in a multiplex similarity search scenario, a plurality of result strands from a single query run can be correlated to a plurality of different queries. Also, the result strand can be of significantly longer length than both the unmatched data strands and the unmatched query strands. Therefore, filtering based on length can provide more accurate results.

Abstract: De novo polynucleotide synthesis is performed with a substrate-bound polymerase. The polymerase is attached to a solid substrate such as a microelectrode array. The polymerase adds nucleotides to growing polynucleotides strands that are also attached to the solid substrate. Spatial control of polymerase activity is achieved by changing the rate of nucleotide polymerization at selected locations on the surface of the solid substrate. The rate of polymerization is changed by inhibiting or promoting activity of the polymerase. In some implementations, activation of electrodes in the microelectrode array changes the rate of nucleotide polymerization. Nucleotides are added to the growing polynucleotide strands at areas where the polymerase is active. By varying the locations where the substrate-bound polymerase is active and the species of nucleotide added, a population of polynucleotides with different, arbitrary sequences is synthesized on the surface of the solid substrate.

Abstract: A method for DNA synthesis using protected nucleosides is disclosed. The nucleosides may be nucleoside triphosphates or nucleoside phosphoramidites with nucleobases attached to electrochemically-cleavable linkers. Removal of a protecting group by application of a voltage in solution triggers a cyclization reaction that cleaves the electrochemically-cleavable linkers. The electrochemically-cleavable linkers may include an amide linkage and an amide that forms a lactam or an ester linkage and a protected alcohol that forms a lactone when the protecting group is removed. The voltage used to cleave the electrochemically-cleavable linkers may be generated by activation of individual electrodes on a microelectrode array. The microelectrode array can be a substrate for solid-phase synthesis of oligonucleotides. Activation of specific electrodes removes the protecting groups at those electrodes and thus enables spatially-controlled extension of the oligonucleotides.

Abstract: A data storage medium is disclosed comprising a substrate covered with alternating layers of a polycationic molecule and artificially synthesized DNA molecules encoding digital information. The magnetic substrate may be a metallic nanoparticle formed from a metal such as iron or cobalt. The polycationic molecule may be polyethyleneimine (PEI). The DNA is protected from degradation by encapsulation in silica. A process for stably storing DNA is also disclosed. Stored DNA may be freed from the silica for sequencing or other analysis by washing the silica-coated DNA with a buffered hydrogen fluoride solution. Storage densities of more than 7% DNA by weight are achieved on nanoparticles.

Abstract: This disclosure describes an efficient method to copy all polynucleotides encoding digital data of digital files in a polynucleotide storage container while maintaining random access capabilities over a collection of files or data items in the container. The disclosure further describes a process whereby random-access and sequencing of the polynucleotides are combined in a single step.

Abstract: Molecular anti-counterfeiting taggants are made from a plurality of synthetic polynucleotides that collectively encode a bit sequence using the sequences and hybridization states of the polynucleotides. The polynucleotide taggant is placed on an item as a molecular identifier of authenticity. The bit sequence encoded by the polynucleotide taggant is read out using a substrate which has bound polynucleotides complexes that hybridize with the synthetic polynucleotides in the polynucleotide taggant. A detectable signal is present where hybridization occurs. To prevent a bad actor from reverse engineering and creating a copy of the polynucleotide taggant using the results of hybridization to the substrate, multiple versions of the substrate are created. Each version hybridizes to different subsets of the synthetic polynucleotides in the polynucleotide taggant. A detectable pattern on the substrate that is present when exposed to the polynucleotide taggant is used for validating authenticity of the item.

Abstract: Multiple polynucleotides with random sequences are collectively used as a molecular anti-counterfeiting tag. The polynucleotides are placed on an item as a molecular identifier of authenticity. Each position within the random sequences is synthesized using a predetermined ratio of nucleoside bases. With this technique the sequence of each polynucleotide is random but the ratio of nucleoside bases over the collection of synthetic polynucleotides is not. Verification of authenticity is achieved by sequencing a portion of the polynucleotides collected from the item and calculating the ratio of nucleoside bases at each position. If these ratios are the same or similar to the ratios used for synthesizing the polynucleotides, then the item is identified as authentic. The ratios of nucleoside bases and a description of the item may be stored in an electronic record that is used for validating authenticity of the item.