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: 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: This disclosure describes particles and techniques for storing oligonucleotides that provide stable, long-term protection yet are also compostable. A core is coated with a layer of oligonucleotides and encapsulated under an outer layer of non-porous, hydrolyzed organosilicon disulfide. The hydrolyzed organosilicon disulfide protects the oligonucleotides from oxidative and thermal damage under typical storage conditions. One suitable organosilicon disulfide is bis(3-triethoxysilylpropyl) disulfide (BTSPD). The oligonucleotides may be retrieved by contacting the particles with a reducing agent that degrades the disulfide bonds in the outer layer. The disulfide bonds enable removal of the protective encapsulation without the use of dangerous chemicals such as hydrogen fluoride. Instead of retrieving the oligonucleotides, the particles may be disposed of in a composting environment. In an implementation, the oligonucleotides are artificially synthesized and encode digital information.

Abstract: This disclosure provides electrochemically-cleavable linkers with cleavage potentials that are less than the redox potential of the solvent in which the linkers are used. In some applications, the solvent may be water or an aqueous buffer solution. The linkers may be used to link a nucleotide to a bound group. The linkers include a cleavable group which may be one of a methoxybenzyl alcohol, an ester, a propargyl thioether, or a trichloroethyl ether. The linkers may be cleaved in solvent by generating an electrode potential that is less than the redox potential of the solvent. In some implementations, an electrode array may be used to generate localized electrode potentials which selectively cleave linkers bound to the activated electrode. Uses for the linkers include attachment of blocking groups to nucleotides in enzymatic oligonucleotide synthesis.

Abstract: High surface area coatings are applied to solid substrates to increase the surface area available for solid-phase synthesis of polymers. The high surface area coatings use three-dimensional space to provide more area for functional groups to bind polymers than an untreated solid substrate. The polymers may be oligonucleotides, polypeptides, or another type of polymer. The solid substrate is a rigid supportive layer made from a material such as glass, a silicon material, a metal material, and plastic. The coating may be thin films, hydrogels, microparticles. The coating may be made from a metal oxide, a high-? dielectric, a low-? dielectric, an etched metal, a carbon material, or an organic polymer. The functional groups may be hydroxyl groups, amine groups, thiolate groups, alkenes, n-alkenes, alkalines, N-Hydroxysuccinimide (NHS)-activated esters, polyaniline, aminosilane groups, silanized oxides, oligothiophenes, and diazonium compounds.

Abstract: Enzymatic polynucleotide synthesis with a template-independent polymerase is used to create multiple polynucleotides having different, arbitrary sequences on the surface of an array. The array provides a spatially-addressable substrate for solid-phase synthesis. Blocking groups are attached to the 3? ends of polynucleotides on the array. Prior to polynucleotide extension, the blocking groups are removed at a selected location on the array. In an implementation, the blocking groups are acyl groups removed with a negative voltage created at an electrode. The array is then incubated with the polymerase and a single species of nucleotide. Nucleotides are incorporated onto the 3? ends of the polynucleotides without blocking groups. Washing removes the polymerase and free nucleotides. To create polynucleotides with different sequences at different locations on the array, the location where the blocking groups are removed and the species of nucleotide may be changed during repeated cycles of synthesis.

Abstract: Multiple polynucleotides having different, arbitrary sequences are synthesized on the surface of an array by spatial control of polymerase activity. The polymerase is a template-independent polymerase such as terminal deoxynucleotidyl transferase (TdT). Spatial control of polymerase activity is implemented by localized changes in redox-pH conditions. A single species of nucleotide is added and incorporated on growing polynucleotide strands at locations on the array where the polymerase is active. A washing step removes the polymerase and free nucleotides. This process may be repeated multiple times changing both the location of polymerase activity and the species of nucleotide thereby synthesizing different polynucleotides in parallel on the surface of the array. Polymerase activity may be regulated by removing a blocking group attached to a His-tag sequence on the polymerase, a change in pH, or release of encapsulated inhibitors.

Abstract: High surface area coatings are applied to solid substrates to increase the surface area available for solid-phase synthesis of polymers. The high surface area coatings use three-dimensional space to provide more area for functional groups to bind polymers than an untreated solid substrate. The polymers may be oligonucleotides, polypeptides, or another type of polymer. The solid substrate is a rigid supportive layer made from a material such as glass, a silicon material, a metal material, and plastic. The coating may be thin films, hydrogels, microparticles. The coating may be made from a metal oxide, a high-? dielectric, a low-? dielectric, an etched metal, a carbon material, or an organic polymer. The functional groups may be hydroxyl groups, amine groups, thiolate groups, alkenes, n-alkenes, alkalines, N-Hydroxysuccinimide (NHS)-activated esters, polyaniline, aminosilane groups, silanized oxides, oligothiophenes, and diazonium compounds.

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 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: 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: Reagents and solvents used for polymer synthesis are reused or recycled rather than discarded. The outflow from each step of polymer synthesis may be collected separately in one of multiple dedicated containers. Reuse returns the outflow from a step of polymer synthesis back to an input of a polymer synthesizer for subsequent use in that same step. Recycling processes the outflow from one or more steps of polymer synthesis to restore original concentrations or purity levels for use in a later synthesis run. Quality control analysis may determine if outflow collected from a polymer synthesizer is reused or recycled. These techniques reduce reagent cost and waste quantity. These techniques may be used with phosphoramidite or enzyme-based synthesis of deoxyribonucleic acid (DNA).

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: 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: High surface area coatings are applied to solid substrates to increase the surface area available for solid-phase synthesis of polymers. The high surface area coatings use three-dimensional space to provide more area for functional groups to bind polymers than an untreated solid substrate. The polymers may be oligonucleotides, polypeptides, or another type of polymer. The solid substrate is a rigid supportive layer made from a material such as glass, a silicon material, a metal material, and plastic. The coating may be thin films, hydrogels, microparticles. The coating may be made from a metal oxide, a high-? dielectric, a low-? dielectric, an etched metal, a carbon material, or an organic polymer. The functional groups may be hydroxyl groups, amine groups, thiolate groups, alkenes, n-alkenes, alkalines, N-Hydroxysuccinimide (NHS)-activated esters, polyaniline, aminosilane groups, silanized oxides, oligothiophenes, and diazonium compounds.

Abstract: Polynucleotide synthesis performed with a substrate independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.

Abstract: Reagents and solvents used for polymer synthesis are reused or recycled rather than discarded. The outflow from each step of polymer synthesis may be collected separately in one of multiple dedicated containers. Reuse returns the outflow from a step of polymer synthesis back to an input of a polymer synthesizer for subsequent use in that same step. Recycling processes the outflow from one or more steps of polymer synthesis to restore original concentrations or purity levels for use in a later synthesis run. Quality control analysis may determine if outflow collected from a polymer synthesizer is reused or recycled. These techniques reduce reagent cost and waste quantity. These techniques may be used with phosphoramidite or enzyme-based synthesis of deoxyribonucleic acid (DNA).

Abstract: The efficiency of polymer synthesis is increased by reducing the number of monomer addition cycles needed to create a set of polymer strands. The number of cycles depends on the sequences of the polymer strands and the order in which each type of monomer is made available for addition to the growing strands. Efficiencies are created by grouping the polymer strands into batches such that all the strands in a batch require a similar number of cycles to synthesize. Efficiencies are also created by selecting an order in which the monomers are made available for addition to the growing polymer strands in a batch. Both techniques can be used together. With these techniques, the number of cycles of monomer addition and commensurate reagent use may be reduced by over 10% as compared to naïve synthesis techniques.

Abstract: A data storage medium is disclosed comprising a dried product formed by drying a salt solution dried together with artificially synthesized DNA molecules encoding digital information. The cation in the salt may be calcium, magnesium, lanthanum, or another cation. The anion in the salt may be chloride, phosphate, or another anion. The DNA is protected from degradation by drying with the salt. Stored DNA may be freed from the salt for sequencing or other analysis by mixing the dried product with a chelator. The dry product formed from DNA and a salt may contain more than 30% DNA by weight and degrade at rates that are less than a third of rate at which untreated DNA degrades.

Abstract: Described herein are systems and methods for coupling environmental hazard detection and actuation of a wearable chemical sensor at a molecular level. The chemical sensor may be a wearable chemical sensor implemented as a powder, cream, lacquer or other wearable construct. The wearable chemical sensor may detect exposure to various environmental hazards and provide an analog means (e.g., a range of color changes) of indicating the level of environmental hazard exposure.